Chitinase chitin-binding fragments

ABSTRACT

The present invention provides chitin-binding fragments of human chitinase, fragment analogs, purified and isolated polynucleotide sequences encoding such fragments and analogs, and materials and methods for the recombinant production of human chitinase fragment products which are expected to be useful as in products for detecting chitin, binding chitin, and treating fungal infections or for development of products useful for treating the same.

[0001] This application is a continuation-in-part of U.S. Ser. No.09/039,198 filed Mar. 12, 1998.

FIELD OF THE INVENTION

[0002] The present invention relates generally to materials comprisingchitin-binding fragments of human chitinase enzyme and analogs of thefragments. More particularly, the invention relates to novel purifiedand isolated polynucleotides encoding such fragment products, to thechitinase fragment products encoded by such polynucleotides, tomaterials and methods for the recombinant production of such chitinasefragment products and to therapeutic and diagnostic uses of suchchitinase fragment products.

BACKGROUND

[0003] Chitin is a linear homopolymer of β-(1,4)-linkedN-acetylglucosamine residues. This polysaccharide is second only tocellulose as the most abundant organic substance. The exoskeleton ofarthropods is composed of chitin. In addition, fungi and other parasitescontain chitin in their outer cell wall, where it serves importantstructural and protective roles. Disruption of the fungal cell wall andmembrane has been a useful therapeutic strategy against fungi andparasites. For example, Amphotericin B and fluconazole exert theiranti-fungal activity by affecting membrane steroids. Despite theexistence of anti-fungal therapeutics, fungal infections of humans haveincreasingly become responsible for life-threatening disorders. See,Georgopapadakou et al., Trends Microbiol., 3: 98-104 (1995). The fungalspecies and parasites responsible for these diseases are mainly Candida,Aspergillus, Cryptococcus, Histoplasma, Coccidioides and Pneumocystis.These pathogens are particularly dangerous in immunocompromisedindividuals, such as patients with AIDS, patients undergoingchemotherapy, and immunosuppressed organ transplant patients.

[0004] Chitin can be degraded by the enzyme chitinase. Chitinase enzymesare found in plants, microorganisms, and animals. Bacterial chitinasehelps to provide a carbon source for bacterial growth. Insects producechitinase to digest their cuticle at each molt. In plants, chitinase isthought to provide a protective role against parasitic fungi. Chitinaseshave been cloned from numerous bacterial [e.g., Serratia marcescens,Jones et al., EMBO J., 5:467-473 (1986)], plant [e.g., tobacco, Heitz etal., Mol. Gen. Genet., 245:246-254 (1994)], and insect [e.g., wasp,Krishnan et al., J. Biol. Chem., 269:20971-20976 (1994)] species andhave been categorized into two distinct families, designated family 18and family 19, based on sequence similarities [Henrissat and Bairoch,Biochem, J. 293:781-788 (1993)]. Although the catalytic region of theenzymes in family 18 is largely conserved across numerous species, thereis very limited sequence similarity across species for thechitin-binding domain. The only feature common to several family 18chitin-binding domains is the presence of multiple cysteine residues.

[0005] Several proteins with low homology to bacterial, insect, andplant chitinases (less than 40% amino acid identity) have beenidentified in mammals, such as human cartilage gp-39 (C-gp39) [Hakala etal., J. Biol. Chem., 268:25803-25810 (1993)], human glycoprotein YKL-40[Johansen et al., Eur. J. Cancer, 31A: 1437-1442 (1995)],oviduct-specific, estrogen-induced protein from sheep [DeSouza et al.,Endocrinology, 136:2485-2496 (1995)], cows and humans; and a secretoryprotein from activated mouse macrophages [Chang et al., Genbank M94584].However, chitin-degrading activity has not been reported for theseproteins. The function of these proteins is not known, but they havebeen postulated to be involved in tissue remodeling. Hakala et al.,supra, report that C-gp39 is detectable in synovial and cartilagespecimens from rheumatoid arthritis patients, but not from normalhumans. Recklies et al., Arthritis Rheumatism, 36(9 SUPPL.):S190 (1993)report localization of the C-gp39 protein to a distinct population ofcells in the superficial layers of cartilage. Johansen et al., supra,report that measurements of YKL-40 serum levels are of value as apotential prognostic marker for the extent of metastatic disease andsurvival of patients with recurrent breast cancer.

[0006] Escott et al., Infect. Immun., 63:4770-4773 (1995) demonstratedchitinase enzymatic activity in human leukocytes and in human serum.Overdijk et al., Glycobiology, 4:797-803 (1994) described isolation of achitinase (4-methylumbelliferyl-tetra-N-acetylchitotetraoside hydrolase)from human serum and rat liver. Renkema et al., J. Biol. Chem.,270:2198-2202 (February 1995) prepared a human chitotriosidase from thespleen of a Gaucher disease patient. Their preparation exhibitedchitinase activity and the article reports a small amount of amino acidsequence of the protein component of the preparation (22 amino terminalresidues and 21 residues of a tryptic fragment). The function of humanchitinase is also unknown, but a relationship with the pathophysiologyof Gaucher disease is proposed in the article. A later publication bythe same group [Boot et al., J. Biol. Chem., 270(44):26252-26256(November 1995)] describes the cloning of a human macrophage cDNAencoding a product that exhibits chitinase activity. The partial aminoacid sequence reported by the group in their February 1995 articlematches portions of the deduced amino acid sequence of the humanmacrophage cDNA product. See also International Patent Publication No.WO 96/40940, which reports two distinct human chitotriosidase cDNAsencoding a 50 kD and a 39 kD product, both of which were fullyenzymatically active. Renkema et al., Eur. J. Biochem., 244:279-285(1997) reported that human chitinase is initially produced inmacrophages as a 50 kD protein that is in part processed into a 39 kDform that accumulates in lysozymes, and also reported that alternativesplicing generates a distinct human chitinase mRNA species encoding a 40kD chitinase. Both the 39 kD and 40 kD isoforms appeared to beC-terminally truncated and displayed full chitinase enzymatic activitybut bound chitin poorly.

[0007] In view of the increasing incidence of life-threatening fungalinfection in immunocompromised individuals, there exists a need in theart to identify new materials and methods useful for diagnosing andtreating fungal infections.

SUMMARY OF THE INVENTION

[0008] The present invention provides novel purified and isolatedpolynucleotides (i.e., DNA and RNA, both sense and antisense strands)encoding human chitinase fragments and analogs thereof havingchitin-binding activity but lacking chitinase enzymatic activity;methods for the recombinant production of such fragment products;purified and isolated human chitinase polypeptide fragment products;pharmaceutical compositions comprising such fragment products; anddiagnostic or therapeutic agents conjugated to such fragment productsthereof. Such fragment products and diagnostic or therapeutic agentsconjugated thereto are expected to be useful for detecting chitin,binding chitin, and treating fungal infections or for development ofproducts useful for treating fungal infections.

[0009] The nucleotide sequence of two human cDNAs encoding presumedallelic variants of human chitinase, and including noncoding 5′ and 3′sequences, are set forth in SEQ ID NO: 1 and SEQ ID NO: 3. The humanchitinase coding region corresponds to nucleotides 2 to 1399 of SEQ IDNO: 1 or nucleotides 27 to 1424 of SEQ ID NO: 3, and the putative codingsequence of the mature, secreted human chitinase protein without itssignal sequence corresponds to nucleotides 65 to 1399 of SEQ ID NO: 1,or nucleotides 90 to 1424 of SEQ ID NO: 3. The amino acid sequences ofthe polypeptides encoded by the DNA of SEQ ID NOS: 1 and 3 are set forthin SEQ ID NO:2 and SEQ ID NO: 4, respectively. Twenty-one amino-terminalamino acids (positions −21 to −1 of SEQ ID NOS: 2 and 4) comprise asignal peptide that is cleaved to yield the mature human chitinaseprotein (positions 1 to 445 of SEQ ID NOS: 2 and 4). It has beendetermined that the seventy-two C-terminal residues of human chitinaseare not critical to chitinase enzymatic activity. Example 5 belowillustrates production of an N-terminal fragment that lacks theseventy-two C-terminal residues of human chitinase; the introduction ofa stop codon after the codon for amino acid 373 resulted in arecombinant chitinase fragment of about 39 kDa that retained similarspecific chitinase enzymatic activity when compared with full lengthrecombinant human chitinase. The cloning of human chitinase cDNA andexpression thereof, and the biological activities of recombinant humanchitinase are described in detail in U.S. application Ser. No.08/877,599 filed Jun. 16, 1997, which is a continuation-in-part of U.S.application Ser. No. 08/663,618 filed Jun. 14, 1996, both of which areincorporated herein by reference in their entirety.

[0010] The present invention is based on the unexpected discovery thatsubstantially all of the chitin-binding activity of human chitinase iscontained within the 99 C-terminal amino acid residues of the 445 aminoacid enzyme. Specifically provided by the present invention arechitin-binding, chitinase-inactive polypeptide products. Preferredchitinase fragment products comprise a chitin-binding fragment withinthe 54 C-terminal amino acids of human chitinase, including a fragmentconsisting of about the 99 C-terminal amino acids of human chitinase(about residues 347 through 445 of SEQ ID NO: 2), a fragment consistingof about the 72 C-terminal amino acids of human chitinase (aboutresidues 374 through 445 of SEQ ID NO: 2), a fragment consisting ofabout the 54 C-terminal amino acids of human chitinase (about residues392 through 445 of SEQ ID NO: 2), and a fragment consisting of about the49 C-terminal amino acids of human chitinase (about residues 397 through445 of SEQ ID NO: 2). Also provided by the invention are purified,isolated polynucleotides including DNA encoding such polypeptidefragments; vectors comprising such DNAs, particularly expression vectorswherein the DNA is operatively linked to an expression control DNAsequence; host cells stably transformed or transfected with such DNAs ina manner allowing the expression in said host cell of human chitinasefragment products; a method for producing human chitinase polypeptidefragment products comprising culturing such host cells in a nutrientmedium and isolating such polypeptides from said host cell or saidnutrient medium; purified, isolated polypeptides produced by thismethod; fusion proteins comprising such polypeptides fused to aheterologous peptide or polypeptide, including an enzyme such assecreted alkaline phosphatase (SEAP); compositions comprising such humanpolypeptide fragment products; compositions comprising a human chitinasepolypeptide fragment product conjugated to an anti-fungal agent andmethods of treating fungal infection by administering such compositions,optionally with co-administration of additional non-chitinaseanti-fungal agents; compositions comprising a chitinase polypeptidefragment product conjugated to a detectable label (includingradioisotopes, fluorophores, dyes, electron-dense compounds andenzymes), methods for using such compositions to determine the presenceor amount of chitin in a sample, comprising the steps of: (a) contactingthe sample with a human chitinase polypeptide fragment productconjugated to a detectable label, and (b) determining the amount oflabelled fragment product bound to chitin, and corresponding kits fordiagnosing the presence of chitin in a sample; monoclonal antibodiesthat specifically bind a chitin-binding, chitinase-inactive fragment ofhuman chitinase, including antibodies that specifically bind to anepitope within the 54 C-terminal amino acids of human chitinase as setforth in SEQ ID NO:2; and preferred monoclonal antibodies 243Q and 243M,and antibodies that compete with or bind to the same epitope as 243Q and243M.

[0011] Chitinase polypeptide fragment products of the invention includefragments of human chitinase or allelic variants thereof thatsubstantially retain chitin-binding activity without retainingsubstantial chitinase enzymatic activity, analogs of such fragments, andfusion proteins comprising such fragments or analogs. Chitinasepolypeptide fragment products are useful in therapeutic and diagnosticapplications as described below.

[0012] Among the “chitin-binding domain” fragments contemplated by theinvention are those represented by amino acid residues X through Y ofSEQ ID NO: 2, wherein X is a consecutive integer from 347 through 397and Y is 445, and portions thereof that retain chitin-binding activity.One preferred fragment consists of the ninety-nine C-terminal aminoacids of human chitinase (residues 347 through 445 of SEQ ID NO: 2);this fragment has been shown in Example 7 below to retain 80% of thechitin-binding activity of the mature chitinase. Yet other preferredfragments are the fifty-four C-terminal amino acids of human chitinase(residues 392 through 445 of SEQ ID NO: 2), and the 49 C-terminal aminoacids of human chitinase (residues 397 through 445 of SEQ ID NO:2),which have also been shown in Example 7 to retain chitin-bindingactivity. As illustrated in Example 7, a fusion protein containing the99 C-terminal amino acids of human chitinase was shown to contain thechitin-binding domain of the protein. The boundaries of thechitin-binding domain were further defined by N-terminal and C-terminaltruncation of this 99 amino acid region and determination of the chitinbinding activity of fusion proteins comprising these truncates. Thesetruncates included those with an N-terminus commencing at amino acidresidue 347, 374, 392, 395, 397, 400 or 409 and with a C-terminus atamino acid residue 431, 443 or 445.

[0013] Analogs may comprise chitinase fragment analogs wherein one ormore of the specified (i.e., naturally encoded) amino acids is deletedor replaced or wherein one or more nonspecified amino acids are added:(1) without loss of one or more of the biological activities (includingchitin-binding activity) or immunological characteristics specific tochitinase; or (2) with specific disablement of a particular biologicalactivity of chitinase. The invention contemplates that conservativeamino acid substitutions as known in the art may be made withoutaffecting the biological activity of the fragment.

[0014] Preferred DNA sequences of the invention include genomic and cDNAsequences as well as wholly or partially chemically synthesized DNAsequences encoding chitin-binding fragments of human chitinase withoutchitinase enzymatic activity, analogs thereof, and fusion proteinscomprising such fragments or analogs. Among the nucleotide sequencescontemplated by the invention are those encoding the amino acidsequences of positions X through Y of SEQ ID NO: 2, wherein X is aconsecutive integer from 347 through 392 and Y is 445. Nucleotides 1238through 1399 of SEQ ID NO: 1 (encoding residues 392 through 445 of SEQID NO: 2) are a particularly preferred DNA sequence of the invention.This DNA sequence and other DNA sequences which hybridize to thenoncoding strand thereof under standard stringent conditions or whichwould hybridize but for the redundancy of the genetic code, and whichencode chitin-binding fragments of a chitinase, are also contemplated bythe invention. Exemplary stringent hybridization conditions are asfollows: hybridization at 42° C. in 50% formamide and washing at 60° C.in 0.1×SSC, 0.1% SDS. It is understood by those of skill in the art thatvariation in these conditions occurs based on the length and GCnucleotide base content of the sequences to be hybridized. Formulasstandard in the art are appropriate for determining exact hybridizationconditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0015] Among the uses for the polynucleotides of the present inventionare use as a hybridization probe, to identify and isolate non-humangenomic DNA and cDNA encoding chitin-binding regions of proteinshomologous to human chitinase; and to identify those cells which expresschitin-binding portions of such proteins and the biological conditionsunder which such proteins are expressed.

[0016] In another aspect, the invention includes biological replicas(i.e., copies of isolated DNA sequences made in vivo or in vitro) of DNAsequences of the invention. Autonomously replicating recombinantconstructions such as plasmid and viral DNA vectors incorporatingpolynucleotides encoding chitin-binding fragments of human chitinase,including any of the DNAs described above, are provided. Preferredvectors include expression vectors in which the incorporated chitinasefragment-encoding cDNA is operatively linked to an endogenous orheterologous expression control sequence and a transcription terminator.Such expression vectors may further include polypeptide-encoding DNAsequences operably linked to the chitinase fragment-encoding DNAsequences, which vectors may be expressed to yield a fusion proteincomprising the polypeptide of interest.

[0017] According to another aspect of the invention, procaryotic oreucaryotic host cells are stably transformed or transfected withpolynucleotide sequences of the invention in a manner allowing thedesired chitinase product to be expressed therein. Host cells expressingchitinase fragment products can serve a variety of useful purposes. Suchcells constitute a valuable source of immunogen for the development ofantibody substances specifically immunoreactive with chitinase. Hostcells of the invention are useful in methods for the large scaleproduction of chitinase fragment products wherein the cells are grown ina suitable culture medium and the desired polypeptide products areisolated, e.g., by immunoaffmity purification, from the cells or fromthe medium in which the cells are grown.

[0018] Knowledge of DNA sequences encoding the chitin-binding portion ofhuman chitinase allows for modification of cells to permit or increaseexpression of the chitin-binding portions. Cells can be modified, (e.g.,by homologous recombination) to provide increased expression of thechitin-binding portion of human chitinase by inserting all or part of aheterologous promoter in the appropriate position within the gene. Theheterologous promoter is inserted in such a manner that it is operablylinked to the DNA sequence encoding the chitin-binding portion of humanchitinase. See, for example, PCT International Publication Nos. WO94/12650, WO 92/20808 and WO 91/09955. Amplifiable marker DNA and/orintron DNA may be inserted along with the heterologous promoter DNA.

[0019] Chitinase fragment products may be obtained as isolates fromnatural cell sources or may be chemically synthesized, but arepreferably produced by recombinant procedures involving procaryotic oreucaryotic host cells of the invention. The use of mammalian host cellsis also expected to provide for post-translational modifications (e.g.,myristolation, glycosylation, truncation, lipidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the invention.

[0020] The invention further comprehends use of chitinase fragmentproducts in screening for proteins or other molecules (e.g., smallmolecules) that specifically bind to the chitin-binding domain of humanchitinase or that modulate binding of human chitinase to chitin or tohuman extracellular matrix proteins such as hyaluronic acid. Proteins orother molecules (e.g., small molecules) which specifically bind tochitinase can be identified using fragments of chitinase isolated fromplasma, recombinant chitinase fragment products, or cells expressingsuch products. Proteins or other molecules that bind to thechitin-binding domain of chitinase may be used to modulate its activity.Binding proteins that specifically bind to the chitin-binding domain ofchitinase are contemplated by the invention and include antibodysubstances (e.g., monoclonal and polyclonal antibodies, single chainantibodies, chimeric antibodies, humanized antibodies, human antibodies,and CDR-grafted antibodies, including compounds which include CDRsequences which specifically recognize a polypeptide of the invention).By “specifically bind to the chitin-binding domain of chitinase” it ismeant that the binding protein recognizes exclusively the chitin-bindingdomain of chitinase and not the catalytically active portion ofchitinase. Binding proteins are useful, in turn, in compositions forimmunization as well as for purifying chitinase, and are useful fordetection or quantification of chitinase in fluid and tissue samples byknown immunological procedures. Anti-idiotypic antibodies specific forchitinase-specific antibody substances are also contemplated.

[0021] Antibodies that specifically bind to chitin-binding domain areuseful in methods for detecting or quantifying the presence ofchitin-binding domain, e.g., in a sandwich ELISA assay, and fordetecting or quantifying the presence of yeast or fungi, e.g., by addinga chitin-binding domain which binds to the yeast or fungi, followed byadding a labeled antibody specific for the chitin-binding domain.Detection of chitin-binding domain in human blood (plasma or serum)samples may also be correlated to a diagnostic standard indicative of adisease state involving chitinase, such as Gaucher's disease. Presentlypreferred antibodies are monoclonal antibodies 243Q and 243M, producedby hybridomas 243Q (Accession No. ______) and 243M (Accession No.______), respectively, and monoclonal antibodies that compete with orbind to the same epitope recognized by 243Q or 243M.

[0022] The scientific value of the information contributed through thedisclosures of DNA and amino acid sequences of the present invention ismanifest. As one series of examples, knowledge of the sequence of a cDNAfor chitinase makes possible the isolation by DNA/DNA hybridization orpolymerase chain reaction (PCR) of genomic DNA sequences encoding othermammalian chitinases and the like. DNA/DNA hybridization or PCRprocedures carried out with DNA sequences of the invention underconditions of stringency standard in the art are likewise expected toallow the isolation of DNAs encoding human allelic variants ofchitinase, other structurally related human proteins sharing thechitin-binding property of chitinase, and the chitin-binding regions ofnon-human species proteins homologous to chitinase. The DNA sequenceinformation provided by the present invention also makes possible thedevelopment, by homologous recombination or “knockout” strategies [see,e.g., Kapecchi, Science, 244: 1288-1292 (1989)], of animals that fail toexpress a functional chitinase enzyme, overexpress chitinase enzyme, orexpress a variant chitinase enzyme. Such animals are useful as modelsfor studying the in vivo activity of chitinase or modulators ofchitinase. Polynucleotides of the invention when suitably labelled areuseful in hybridization assays to detect the capacity of cells tosynthesize chitinase. Polynucleotides of the invention may also be thebasis for diagnostic methods useful for identifying a geneticalteration(s) in the chitinase locus that underlies a disease state orstates. Also made available by the invention are anti-sensepolynucleotides relevant to regulating expression of chitinase by thosecells which ordinarily express the same.

[0023] The invention contemplates that chitin-binding fragment productsmay be fused to a heterologous polypeptide. For example, such productsmay be fused to a portion of an immunoglobulin, such as the constantregion, for therapeutic purposes. As another example, such products maybe fused to a polypeptide useful as a detectable label or marker, suchas a polypeptide with enzymatic activity or a polypeptide carrying aspecifically detectable epitope, such as a myc epitope or FLAG epitopetag (Eastman Kodak).

[0024] Chitin-binding fragments may also be fused to another protein ofinterest to facilitate purification of the protein of interest viaaffinity binding to a chitin matrix. The fusion protein may then beobtained by elution from the column, or the protein of interest may becleaved from the chitin-binding domain followed by elution of thecleaved protein. See Chong et al., Gene, 192:271-281 (1997).

[0025] The human chitinase fragment products of the invention are alsouseful as a chitin-specific reagent for specifically identifying thepresence of chitin in a sample. According to this aspect of theinvention, a chitinase fragment product having chitin-binding activityis conjugated with a detectable label, such as a radioisotope,fluorophore, dye, electron-dense compound, or enzyme, contacted with thesample to be tested, and analyzed qualitatively or quantitatively forthe presence of chitin. “Conjugated” as used herein means linked bycovalent bonds. Such techniques are well known and illustrated in, e.g.,U.S. Pat. No. 5,587,292, incorporated herein by reference. The amount ofchitin thus measured can be indicative of the fungal load in an infectedpatient. Two preferred fragments for use according to this method arethe 54 amino acid chitin-binding domain consisting of amino acidresidues 392 through 445 of the human chitinase amino acid sequence setout in SEQ ID NO: 2 and the 49 amino acid chitin-binding domainconsisting of amino acid residues 397 through 445 of SEQ ID NO: 2.

[0026] The invention also provides conjugates comprising chitin-bindingchitinase fragments and an imaging agent, such as gamma- andpositron-emitting radioisotopes for radionuclear imaging (e.g., 157Gd,55Mn, 162Dy, 52Cr, 56Fe, 111In, 97Ru, 67Ga, 68Ga, 72As, 89Zr, 201Tl,99Tn, 90Y); paramagnetic metal chelates, nitroxyl spin labelledcompounds or other agents (e.g., Gd(III), Eu(III), Dy(III), Pr(III),Pa(IV), Mn(II), Cr(III), Co(III), Fe(III), Cu(II), Ni(II), Ti(III) andV(IV), GdDTPA/dimeglumine [Magnevist™]) for MRI imaging; contrastenhancement agents for X-ray based imaging, including CT scans (e.g.,bromine- or iodine-containing compounds); and other agents known in theart. Such conjugates are expected to bind yeast cell wall chitin andthus to be useful in methods for detecting or localizing yeast in vivoin mammals.

[0027] Administration of chitinase fragment products and therapeuticagents comprising such products to mammalian subjects, especiallyhumans, for the purpose of ameliorating disease states caused bychitin-containing parasites such as fungi is contemplated by theinvention. Fungal infections (mycoses) such as candidiasis,aspergillosis, coccidioidomycosis, blastomycosis,paracoccidioidomycosis, histoplasmosis, cryptococcosis,chromoblastomycosis, sporotrichosis, mucormycosis, and thedermatophytoses can manifest as acute or chronic disease. Pathogenicfungi cause serious, often fatal disease in immunocompromised hosts.Cancer patients undergoing chemotherapy, immunosuppressed individuals,and HIV-infected individuals are susceptible to mycoses caused byCandida, Aspergillus, Pneumocystis carinii, and other fungi.Amphotericin B and fluconazole are useful therapeutics for fungalinfections, but toxicity associated with these drugs causes seriousadverse side effects that limit their usefulness. The mortality ofsystemic candidiasis is greater than 50% despite Amphotericin Btreatment. Animal models for fungal infection are illustrated below inExamples 9 through 15 and have been described in the art.

[0028] Specifically contemplated by the invention are compositionscomprising chitinase fragment products for use in methods for treating amammal susceptible to or suffering from fungal infections. It iscontemplated that the chitinase fragment products may be conjugated toother conventional anti-fungal agents, including amphotericin B and thestructurally related compounds nystatin and pimaricin; 5-fluorocytosine;azole derivatives such as fluconazole, ketoconazole, clotrimazole,miconazole, econazole, butoconazole, oxiconazole, sulconazole,terconazole, itraconazole and tioconazole; allylamines-thiocarbamates,such as tolnaftate, naftifine and terbinafine; griseofulvin; ciclopiroxolamine; haloprogin; undecylenic acid; and benzoic acid. [See, e.g.,Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed.,McGraw-Hill, NY (1996).] According to this aspect of the invention, thechitin-binding fragment products serve as a vector to target knownfungicidal or fungistatic compounds to pathogenic chitin-bearing fungi,and thus may improve the effectiveness of these conventional anti-fungalagents, perhaps by rendering the fungi more susceptible to their action.A reduction in the amount of conventional anti-fungal agent needed toexert the desired therapeutic effect may allow the drugs to be used atless toxic levels. The ability to selectively target fungi or yeastusing a chitin-binding domain fragment also allows administration ofsuch fragments conjugated to cytotoxic agents that are not themselvesselectively anti-fungal. This aspect of the invention contemplatesconjugation of chitin-binding chitinase fragments to any cytotoxic agentknown in the art, including radioisotopes (such as 90Y, 188Re, 186Re,199Au, 64Cu, 67Cu, 131I), toxins and chemotherapeutic agents, that wouldbe effective against yeast. Suitable cytotoxic agents can be easilyidentified using methods known in the art. Using human chitinasechitin-binding domain for this purpose is more advantageous than usingchitin-binding domains of chitinases of other species because humanpolypeptides are expected to be non-immunogenic in humans.

[0029] Chitin-binding domain fragments themselves may have anti-fungaleffects through disruptive cross-linking of yeast cell wall, and may beco-administered alone or in combination with other anti-fungal agents.Multimeric chitin-binding domain fragments, which may be especiallyuseful for this purpose, are contemplated by the invention, includingmultimeric fragments that have been covalently cross-linked by chemicalmeans and recombinantly produced polypeptides comprising multiplechitin-binding domains linked in tandem. The administration ofchitin-binding domain fragments, either monomeric or multimeric, mayreduce the amount of co-administered anti-fungal agent necessary toexert a desired therapeutic effect.

[0030] Thus, the invention contemplates the use of chitinase fragmentproducts in the preparation of a medicament for the prophylactic ortherapeutic treatment of fungal infections.

[0031] Therapeutic/pharmaceutical compositions contemplated by theinvention include chitinase fragment products, which may be conjugatedto another therapeutic agent, and a physiologically acceptable diluentor carrier and may also include other anti-fungal agents. Dosage amountsindicated would be sufficient to supplement endogenous chitinaseactivity. For general dosage considerations see Remington: The Scienceand Practice of Pharmacy, 19th ed., Mack Publishing Co., Easton, Pa.(1995). Dosages will vary between about 1 μg/kg to 100 mg/kg bodyweight, and preferably between about 0.1 to about 20 mg chitinase/kgbody weight. Therapeutic compositions of the invention may beadministered by various routes depending on the infection to be treated,including via subcutaneous, intramuscular, intravenous, intrapulmonary,transdermal, intrathecal, topical, oral, or suppository administration.

[0032] The invention also contemplates that the overexpression ofchitinase in Gaucher disease or at sites of inflammation (such as inrheumatoid arthritis) may have deleterious effects on the extracellularmatrix and, in such disease settings, inhibitors of chitinase activity,including chitinase fragment products themselves or inhibitors ofchitin-binding identified by the screening methods described above, mayprovide therapeutic benefit, e.g. by reducing remodeling or destructionof the extracellular matrix.

[0033] The human chitinase cDNA has been isolated from a macrophage cDNAlibrary. Macrophages are known to be closely associated with rheumatoidarthritis lesions [Feldman et al., Cell, 85:307-310 (1996)], andmacrophage products such as TNF-α are implicated in disease progression.A protein with homology to human chitinase, C-gp39, has been detected inthe synovium and cartilage of rheumatoid arthritis patients. While thenatural substrate for human chitinase is probably chitin from pathogenicorganisms, the enzyme may also exhibit activity on endogenousmacromolecules which form the natural extracellular matrix. For example,it has been suggested that hyaluronic acid, a major component of theextracellular matrix, contains a core of chitin oligomers. [Semino etal., Proc. Nat'l Acad. Sci., 93:4548-4553 (1996); Varki, Proc. Nat'l.Acad. Sci., 93:4523-4525 (1996).] Chitinase may therefore be involved indegradation of extracellular matrix in diseases such as rheumatoidarthritis. The role of chitinase may be determined by measuringchitinase levels and/or the effects of chitinase administration orchitinase inhibition in synovial fluid isolated from arthritic joints.Endogenous chitinase levels can be measured by enzymatic assay or withan antibody. Viscosity of synovial fluid can be measured before andafter chitinase treatment; a decrease of viscosity associated withchitinase would be consistent with an endogenous chitinase substrate.Modulation of chitinase activity could thereby modulate the progressionof joint destruction in rheumatoid arthritis.

[0034] Also contemplated by the invention are methods for screening forinhibitors of chitinase activity, which may be useful in the mannerdescribed in the preceding paragraph. A method for screening samples toidentify agents that inhibit chitinase is reported in, e.g., WO 95/34678published Dec. 21, 1995.

DETAILED DESCRIPTION OF THE INVENTION

[0035] Other aspects and advantages of the present invention will beunderstood upon consideration of the following illustrative examples.Example 1 describes the isolation of human chitinase cDNA clones from ahuman macrophage cDNA library. Example 2 addresses the pattern ofchitinase gene expression in various human tissues. Example 3 describesthe recombinant expression of the human chitinase gene in prokaryoticcells and purification of the resulting enzyme. Example 4 provides aprotocol for the recombinant production of human chitinase in yeast.Example 5 describes the recombinant expression of the human chitinasegene in mammalian cells and purification of the resulting protein.Example 6 describes production of human chitinase polypeptide analogsand fragments by peptide synthesis or recombinant production methods.Example 7 describes production of human chitinase fragments havingchitin-binding activity and analogs thereof. Example 8 provides aprotocol for generating monoclonal antibodies that are specificallyimmunoreactive with human chitinase. Example 9 describes an assay forthe measurement of chitinase catalytic activity. Example 10 addressesdetermination of the anti-fungal activity of test drugs in vitro.Example 11 addresses determination of the anti-fungal activity of testdrugs in vivo in a mouse model, and Examples 12 through 15 addressrabbit models of invasive aspergillosis, disseminated candidiasis,Candida ophthalmitis, and Candida endocarditis. Example 16 compareschitin-binding and chitin hydrolysis activities of full length humanchitinase and a C-terminally truncated fragment. Example 17 addressesconjugation of chitin-binding fragments to other moieties.

EXAMPLE 1 Isolation of Chitinase cDNA Clones

[0036] A cDNA library was prepared from peripheral bloodmonocyte-derived macrophages as described in Tjoelker et al., Nature,374:549-552 (1995). Clones from the library were randomly chosen andplasmid DNA was purified from individual clones. The sequence ofapproximately 300 to 500 bases from the end of DNA from each clone wasdetermined on an automated sequencer (Model 373, Applied Biosystems,Foster City, Calif.) using primer JHSP6, which hybridizes to the plasmidvector pRc/CMV (Invitrogen, San Diego, Calif.) adjacent to the cDNAcloning site:

[0037] JHSP6: 5′-GACACTATAGAATAGGGC-3′ (SEQ ID NO: 5)

[0038] The nucleotide and deduced amino acid sequence of these cDNAclones were compared to sequences in nucleotide and peptide sequencedatabases to determine similarity to known genes. Sequence comparisonswere performed by the BLAST Network Service of the National Center forBiotechnology Information using the alignment algorithm of Altschul etal., J. Mol. Biol., 215:403-410 (1990). Clone MO-91 1 exhibitedsignificant homology to several different sequences, including mousemacrophage secretory protein YM-1 precursor (Genbank accession no.M94584), human cartilage gp-39 (Hakala et al., supra), oviductalglycoprotein from sheep, cow, and humans (DeSouza et al., supra), andchitinases from parasite (Oncocerca, Genbank accession no. U14639), wasp(Chelonus, Genbank accession no. U10422), plant (Nicotiana, Genbankaccession no. X77111), and bacteria (Serratia, Genbank accession no.Z36295); its highest observed homology was to mammalian genes thatencoded proteins with chitinase homology but no demonstrated chitinaseactivity. Further sequence analysis of MO-911 suggested that itcontained a portion of the coding region for a human chitinase homolog.

[0039] The DNA sequence of clone pMO-218 (deposited on Jun. 7, 1996under the terms of the Budapest Treaty with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. underAccession No. 98077) is set forth in SEQ ID NO: 1, and the encoded aminoacid sequence is set forth in SEQ ID NO: 2. MO-218 appeared to includethe entire coding region of the human chitinase cDNA (nucleotides 2 to1399 of SEQ ID NO: 1), which comprises a twenty-one amino acid putativesignal sequence followed by 445 encoded amino acids (residues 1 to 445of SEQ ID NO: 2). The twenty-two amino acids following the putativesignal sequence exactly match the amino-terminal sequence of purifiedhuman chitotriosidase reported in Renkema et al., supra. Renkema et al.also described a twenty-one amino acid sequence from a tryptic fragmentof human chitotriosidase which corresponds exactly to residues 157 to177 of MO-218 (SEQ ID NO: 2). Boot et al., supra, report the cloning ofa human chitotriosidase cDNA which contains a coding sequenceessentially identical to that of MO-218. The sequence of MO-218 differsfrom Boot et al. by an additional fourteen nucleotides at the 5′ end andby a nucleotide change at nucleotide 330 in the coding region.

[0040] To confirm that MO-218 indeed contained the entire coding regionof the cDNA, a ³²P-labelled probe P-1(TGGGATCATCAGCAGGACCATGAAACCTGCCCAGGCCACAGACCGCACCAT, SEQ ID NO: 6) wasprepared that corresponded to the complement of nucleotides 2 through 52of MO-218 (SEQ ID NO: 1). Probe P-1 was designed to hybridize withclones that are at least as long as MO-218 at the 5′ end. The probe washybridized with a portion (approximately 30,000 clones) of the humanmacrophage cDNA library described above, in 40% formamide andhybridization buffer (5×SSPE, 10×Denhardt's, 100 μg/ml denatured salmonsperm DNA, and 2% SDS) at 42° C. overnight. The filters were washed andthree clones that hybridized were chosen for sequence analysis. Thelongest clone was designated pMO-13B (deposited on Jun. 7, 1996 underthe terms of the Budapest Treaty with the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, U.S.A. underAccession No. 98078). The DNA sequence of pMO-13B is set forth in SEQ IDNO: 3 and the encoded amino acid sequence is set forth in SEQ ID NO: 4.This clone contains 25 additional nucleotides at the 5′ end comparedwith MO-218; in addition, MO-13B (SEQ ID NO: 3) contains one nucleotidesubstitution at nucleotide 330 (corresponding to nucleotide 305 ofMO-218, SEQ ID NO: 1) which changes the encoded amino acid at position80 of the mature protein from a glycine (in SEQ ID NO: 2) to a serine(in SEQ ID NO: 4).

EXAMPLE 2 Chitinase Gene Expression Pattern in Human Tissues

[0041] Northern blot analysis was performed to identify tissues in whichthe human chitinase is expressed. A multiple human tissue Northern blot(Clontech, Palo Alto Calif.) was hybridized with the entire codingregion of MO-218 under standard stringent conditions (according to theClontech laboratory manual). Greatest hybridization was observed to lungtissue (+++) and ovary (+++), with much smaller levels (+) in thymus andplacenta. The size of the hybridizing mRNA was 2.0 kb for lung, ovaryand thymus, which corresponds well with the size of the cloned cDNA (1.6kb, or about 1.8 kb including the polyA tail). The size of thehybridizing placental mRNA was considerably smaller, at 1.3 kb.Chitinase hybridization was not observed in spleen, prostate, testes,small intestine, colon, peripheral blood leukocytes, heart, brain,liver, skeletal muscle, kidney, or pancreas. Chitinase expression inlung is consistent with a protective role against pathogenic organismsthat contain chitin, since the lung represents the primary route ofentry for fungal pathogens.

EXAMPLE 3 Production of Recombinant Human Chitinase in Bacterial Cells

[0042] The mature coding region of MO-218 was engineered for expressionin E. coil as a C-terminal truncated analog. PCR was used to generate aDNA fragment for expression using a primer corresponding to nucleotides65 to 88 of the MO-218 chitinase cDNA preceded by an initiatingmethionine codon and an XbaI restriction endonuclease site(5′-TACATCTAGAATTATGGCAAAACTGGTCTGCTACTTCACC-3′, SEQ ID NO: 7), and adownstream primer encoding nucleotides 1163 to 1183 of MO-218 followedby a stop codon and a HindIII site(5′-AGATCTAACCTTAGGTGCCTGAAGACAAGTATGG-3′, SEQ ID NO: 8). The downstreamprimer contained an adenine at base 25, while the MO-218 sequencecontains a guanine at the corresponding nucleotide position.Consequently, the resulting DNA fragment contains a thymine rather thana cytosine at the position corresponding to nucleotide 1172 of theMO-218 sequence, and the encoded chitinase fragment, set forth in SEQ IDNO: 15, is also an analog that contains a serine at mature amino acidposition 370 instead of the proline encoded by MO-218. The resulting DNAfragment was digested with XbaI and HindIII and cloned into plasmidpAraBAD (which is also known by the designation pAraCB).

[0043] Plasmid pAraCB was prepared as follows. Plasmid pUC19 wasmodified to include an arabinose promoter and subsequently to includeAKAP 79 encoding sequences. The arabinose promoter [Wilcox et al., Gene,34:123-128 (1985); Wilcox, et al., Gene, 18:157-163 (1982)] and the araCgene were amplified by PCR from the arabinose operon BAD of Salmonellatyphimurium as an EcoRI/XbaI fragment with the primers araC-2 (SEQ IDNO: 9) and arab-1 (SEQ ID NO: 10): araC-2 TACAGAATTC TTATTCACATCCGGCCCTGSEQ ID NO:9 arab-1 TACATCTAGA CTCCACTCCATCCAGAAAAACAGGTATGG SEQ ID NO:10

[0044] Primer araC-2 encodes an EcoRI site (underlined) and atermination codon (italics) for the araC gene product. Primer arab-1encodes a putative ribosome binding domain (italics) and an XbaIrestriction site (underlined). PCR with these primers produced a 1.2 kbfragment which was digested with EcoRI and XbaI and subcloned into pUC19(New England Biolabs, Beverly, Mass.) previously digested with the sametwo enzymes. The resulting plasmid was designated pAraCB and contained apolylinker region (SEQ ID NO: 11) flanked at the 5′ end with a XbaIrestriction site (underlined) and at the 3′ end with a HindIII site(italics).

[0045] araCB polylinker TCTAGAGTCGACCTGCAGGCATGCAAGCTT SEQ ID NO: 11

[0046] Transformants containing the resulting expression plasmid(pAraMO218) were induced with arabinose and grown at 37° C. Thesetransformants produced inclusion bodies containing a 39 kDa proteinwhich was a truncated form of chitinase (engineered to contain 373instead of 445 amino acids). This chitinase fragment contains fourcysteine residues, while the full length chitinase contains ten cysteineresidues. The inclusion bodies were separated from the E. coli cultureand electrophoresed on SDS-PAGE. The 39 kDa band was transferred to aPVDF membrane and amino terminal sequenced. The majority (abouttwo-thirds) of the material contained a sequence corresponding to theamino terminus of human chitinase. The remaining material correspondedto a contaminating E. coli protein, porin. This recombinant chitinasepreparation from E. coli was useful for producing polyclonal andmonoclonal antibodies (described below in Example 8).

[0047] When transformants containing the Ara-chitinase expressionplasmid were grown at 25° C., inclusion bodies were not observed andexpression of recombinant product was decreased from about ten percentof total cell protein to about one percent. However, this materialproduced at 25° C. exhibited chitinase catalytic activity.

EXAMPLE 4 Production of Recombinant Human Chitinase in Yeast Cells

[0048] Exemplary protocols for the recombinant expression of humanchitinase in yeast and for the purification of the resulting recombinantprotein follow. The coding region of human chitinase is engineered intovectors for expression in Saccharomyces cerevisiae using either PCR orlinker oligonucleotides designed to encode a fusion polypeptidecontaining a secretion mediating leader to the coding region for humanchitinase corresponding to the amino terminus of the natural molecule.Secretion signal peptides include, e.g., SUC2 or equivalent leaders witha functional signal peptidase cleavage site, or pre-pro-alpha factor orother complex leader composed of a pre, or signal peptide, and a pro, orspacer region, exhibiting a KEX2 cleavage site. The DNA encoding thesignal sequence can be obtained by oligonucleotide synthesis or by PCR.The DNA encoding the pre-pro-alpha factor leader is obtained by PCRusing primers containing nucleotides 1 through 20 of the alpha matingfactor gene and a primer complementary to nucleotides 255 through 235 ofthis gene [Kurjan and Herskowitz, Cell, 30:933-943 (1982)]. Thepre-pro-alpha leader coding sequence and human chitinase coding sequencefragments are ligated into a plasmid containing the yeast alcoholdehydrogenase (ADH2) promoter, such that the promoter directs theexpression of a fusion protein. As taught by Rose and Broach, [Meth.Enz., 185:234-279, D. Goeddel, ed., Academic Press, Inc., San Diego,Calif. (1990)], the vector further includes an ADH2 transcriptionterminator downstream of the cloning site, the yeast “2-micron”replication origin, a selectable marker, for example TRP1, CUP1 or LEU2(or LEU2-d) or other equivalent gene, the yeast REP1 and REP2 genes, theE. coli beta lactamase gene, and an E. coli origin of replication. Thebeta-lactamase and TRP1 genes provide for selection in bacteria andyeast, respectively. The REP1 and REP2 genes encode proteins involved inplasmid copy number replication.

[0049] Alternatively, other fusion points within the chitinase codingregion may be chosen. Truncates of the coding region may be used toincrease homogeneity of the product, increase the specific activity oralter the substrate specificity.

[0050] The DNA constructs described in the preceding paragraphs aretransformed into yeast cells using a known method, e.g. lithium acetatetreatment [Steams et al., Meth. Enz., supra, pp. 280-297] or byequivalent methods. The ADH2 promoter is induced upon exhaustion ofglucose in the growth media [Price et al., Gene, 55:287 (1987)]. Thepre-pro-alpha sequence or other leader sequence effects secretion of thefusion protein, releasing the mature human chitinase peptide from thecells. The signal peptide leader is processed by signal peptidase or, inthe case of pre-pro-alpha removal of the pro region, by the KEX2protease [Bitter et al., Proc. Natl. Acad. Sci. USA, 81:5330-5334(1984)].

[0051] Chitinase contains in its mature amino acid sequence two dibasicsequences at positions 107-108 (Lys-Arg) and 209-210 (Arg-Lys) that maybe proteolytically clipped by the KEX2 protease during secretion. Tostabilize and/or increase the level of product secreted from cells,these sequences could be mutated to eliminate the potential sites forproteolysis as shown by Gillis et al. [Behring Inst. Mitt., No. 83:1-7(1988)] or by expressing chitinase without dibasic modifications in ahost that is deficient in KEX2. Such hosts can be obtained either byscreening for non-KEX2 protease containing mutants, or by manipulationof the genomic KEX2 locus by gene replacement/gene disruption techniques[Orr-Weaver et al., Proc. Natl. Acad. Sci, USA, 78:6354-6358 (1981)].

[0052] Recombinant chitinase may be secreted from Saccharomycescerevisiae using similar vectors containing alternative promoters PRB1,GAL4, TPI, or other suitably strong promoters bearing fragments or byfusion to a variety of leader sequences [Sleep et al., Bio/Technol.,8:42-46 (1990)].

[0053] Other non-Saccharomyces cerevisiae suitable expression hostsinclude Kluyveromyces lactis, Schizosaccharomyces pombe, Pichia pastorisand members of the Hansenula or Aspergillus geni. Analogous recombinantexpression systems for these fungi include the organism and theirappropriate autonomously replicating vector [e.g. Falcone et al.,Plasmid, 15:248-252 (1988)] or multiply integrated expression cassettes.These systems also rely on signal sequences or leaders of the typesdescribed above to mediate secretion into the medium.

[0054] The secreted recombinant human chitinase is purified from theyeast growth medium by, e.g., the methods used to purify chitinase frombacterial and mammalian cell supernatants (see Example 3 above andExample 5 below).

[0055] Alternatively, the mature form of the recombinant chitinaseproduct may be expressed in the cytoplasms of the Saccharomycescerevisiae cells or analogous host, and purified from the lysed hostcells. The protein may be refolded during the act of purification toobtain appropriate levels of specific activity.

EXAMPLE 5 Production of Recombinant Human Chitinase in Mammalian cells

[0056] A. Expression in COS Cells

[0057] The MO-218 clone and the MO-13B clone, both of which contain fulllength human chitinase cDNA 3′ to the CMV promoter of pRc/CMV, wereisolated. A third plasmid, which corresponded to the same C-terminalfragment expressed in bacterial cells in Example 3 above, was preparedas follows. The MO-218 plasmid was amplified by PCR usingoligonucleotide primer 218-1(CGCAAGCTTGAGAGCTCCGTTCCGCCACATGGTGCGGTCTGTGGCCTGGG, SEQ ID NO: 12),which contains a Hind III site and nucleotides 2 through 23 of theMO-218 chitinase cDNA of SEQ ID NO: 1, and with complementary downstreamprimer T-END (GACTCTAGACTAGGTGCCTGAAGGCAAGTATG, SEQ ID NO: 13), whichcontains nucleotides 1164 through 1183 of MO-218, a stop codon and anXbaI site. The amplified DNA was purified by electrophoresis, digestedwith XbaI and HindIII, and cloned into the pRc/CMV vector (Invitrogen,San Diego, Calif.) previously cut with the same restriction enzymes. Thejunctions of the resulting clone was sequenced on a Model 373 (AppliedBiosystems, Foster City, Calif.), confirming that the clone encoded thepredicted engineered protein sequence, set forth in SEQ ID NO: 14.

[0058] All three plasmids were transiently transfected into COS cells bythe DEAE transfection method [see, e.g., Sambrook et al., MolecularCloning: a Laboratory Manual, 2d ed., Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory (1989).). After three days at 37° C., mediafrom cells was assayed for chitinase activity in vitro as describedbelow in Example 9. Each culture produced significant chitinase activity(600-800 mU/ml/min), and similar amounts were observed for eachconstruct.

[0059] Recombinant human chitinase was purified as follows. Five daysafter transfection of COS cells with the pRc/CMV-MO-13B plasmid,conditioned media from the culture was harvested and diluted with anequal volume of water. The diluted conditioned media was applied to aQ-Sepharose Fast Flow column (Pharmacia Biotech, Uppsala, Sweden) whichwas pre-equilibrated in 25 mM Tris, 10 mM sodium chloride, 1 mM EDTA, atpH 8.0. Approximately 95% of the chitinase activity flowed through anddid not bind to the column. This Q-Sepharose flow through was adjustedto 1.2 M ammonium sulfate and applied to a Butyl-Sepharose 4 Fast Flowcolumn (Pharmacia) which was pre-equilibrated in 25 mM Tris, 1.2 Mammonium sulfate, 1 mM EDTA, at pH 8.0. Protein was eluted using areverse gradient of 1.2 M to 0 M ammonium sulfate in 25 mM Tris, pH 8.0.A single absorbance peak at 280 nm corresponding to the chitinaseactivity peak was eluted at low salt. This material was greater than 85%pure as determined by SDS-PAGE and contained approximately 60% of thechitinase activity. The protein was then concentrated and bufferexchanged into 20 mM Tris, 150 mM sodium chloride, at pH 8.0 using a10,000 MWCO membrane (Ultrafree™ 10K, Millipore Corp., Bedford, Mass.).This preparation was then tested for enzymatic and anti-fungal activityin vitro as described in Examples 9 and 10 below. The recombinantpreparation had a chitotriosidase activity of 90 nmol/min per mgprotein.

[0060] B. Expression in CHO Cells

[0061] The chitinase gene was inserted into pDEF1 (the construction ofwhich is described in Example 4 of U.S. application Ser. No. 08/847,218filed May 1, 1997, incorporated herein by reference) by excising the1.77 kb HindIII/XbaI fragment containing the full length chitinase genefrom pRc/CMV/MO-13B and ligating the fragment with HindIII/XbaI-digestedpDEF1, to create plasmid pDEF1/CTN.1. The 1.77 kb HindIII/XbaI fragmentcontaining the chitinase gene was also ligated intoHindIII/XbaI-digested pHDEF1 to create plasmid pHDEF1/CTN.1. PlasmidpHDEF1 is the same as pDEF1 except for two differences: (1) in pHDEF1, a2 kb EheI/SalI fragment derived from pCEP4 (Invitrogen, Carlsbad,Calif.) containing a hygromycin resistance gene replaced the 19 bpPmeI/SalI fragment of pDEF1; (2) in pHDEF1, expression of thedihydrofolate reductase (DHFR) gene is controlled by a shortened SV40promoter contained on a 120 bp NheI/Asp718 fragment that replaced thecorresponding 212 bp NheI/Asp718 fragment of pDEF1. This 120 bpNheI/Asp718 fragment was prepared by first amplifying a 171 bp PCRfragment with oligonucleotide primer 94-26(5′-TGATACGGTACCGCCCCATGGCTGACTA-3′, SEQ ID NO: 16) (which contains anew Asp718 site), and primer 94-27 (5′-GCAAGTTTGGCGCGAAATCG-3′, SEQ IDNO: 17), using as a template the DNA from pDC1 (described in Example 4of U.S. application Ser. No. 08/847,218 filed May 1, 1997) that carriesthe SV40-DHFR cassette, and then digesting this 171 bp PCR fragment withNheI and Asp718.

[0062] The DHFR-negative Chinese hamster ovary (CHO) cell line DG44 wastransfected with plasmid pDEF1/CTN.1 as described in Example 5 of U.S.application Ser. No. 08/847,218 filed May 1, 1997. The CHO cell lineDG44 was also transfected with plasmid pHDEF1/CTN.1, followed byselection using the following modified procedure. The cells were firstselected for hygromycin resistance only, in media (DMEM/F-12supplemented with 2-10% dialyzed FBS) containing 800 mg/liter ofhygromycin (Calbiochem, San Diego, Calif.) and also containinghypoxanthine and thymidine (which therefore made the media non-selectivefor the DHFR gene). After selecting transfectants that were resistant tohygromycin, the cells were further selected for expression of the DHFRgene by growing them in media lacking hypoxanthine and thymidine. Next,the DHFR-positive and hygromycin-resistant CHO cells were selected inmedia containing first 10 nM, then 20 nM, and finally 50 nMmethotrexate, which resulted in selection of cells expressing higherlevels of chitinase.

[0063] The supernatant from the pHDEF1/CTN.1 transfected CHO cellscontaining overexpressed recombinant human chitinase (rH-Chitinase) waspurified as follows. In preparation for anion exchange chromatography,the supernatant was diluted 1:3 with 20 mM Tris, pH 7.0 (Buffer A). Ananion exchange column, packed with Q-Sepharose Fast Flow Resin(Pharmacia Biotech Inc., Piscataway, N.J.), was equilibrated with BufferA and loaded with 15 L diluted supernatant per 1 L resin. TherH-Chitinase was collected in the Flow Through from the Q-Sepharosecolumn and adjusted to 5% Polyethylene Glycol (PEG) 400 (MallinckrodtBaker, Inc., Phillipsburg, N.J.), 30 mM sodium acetate, pH 4.3 inpreparation for cation exchange chromatography. A cation exchangecolumn, packed with CM-Sepharose Fast Flow Resin (Pharmacia BiotechInc., Piscataway, N.J.), was equilibrated with 30 mM sodium acetate, 5%PEG 400, pH 4.3 (Buffer B). The rH-Chitinase sample was loaded onto theCM-Sepharose column at 1 mg per mL resin, and rH-Chitinase was elutedfrom the column with 40 mM Tris, 5% PEG 400, pH 7.5 (Buffer C). TherH-Chitinase sample was then prepared for hydrophobic interactionchromatography by adding (NH₄)₂SO₄ to 1.5 M. A column packed withMacro-Prep Methyl H1C Support, (Bio-Rad Laboratories, Hercules, Calif.,)was equilibrated with 20 mM Tris, 5% PEG 400, pH 7.0 (Buffer D)containing 1.5 M (NH₄)₂SO₄. The rH-Chitinase sample was loaded onto theMacro-Prep Methyl column at 1 mg per mL resin. The column was washedwith Buffer D containing 1.1 M (NH₄)₂SO₄, and rH-Chitinase was elutedwith Buffer D containing 0.2 M (NH₄)₂SO₄. The purified eluate wasexchanged into 150 mM NaCl, 20 mM Tris, pH 7.0 (Buffer E) by membranefiltration.

EXAMPLE 6 Production of Human Chitinase Analogs and Fragments

[0064] Recombinant techniques such as those described in the precedingexamples may be used to prepare human chitinase polypeptide analogs orfragments. More particularly, polynucleotides encoding human chitinaseare modified to encode polypeptide analogs of interest using well-knowntechniques, e.g., site-directed mutagenesis and polymerase chainreaction. C-terminal and N-terminal deletions are also prepared by,e.g., deleting the appropriate portion of the polynucleotide codingsequence. See generally Sambrook et al., supra, Chapter 15. The modifiedpolynucleotides are expressed recombinantly, and the recombinantpolypeptide analogs or fragments are purified as described in thepreceding examples.

[0065] Residues critical for human chitinase activity are identified,e.g., by homology to other chitinases and by substituting alanines forthe native human chitinase amino acid residues. Cysteines are oftencritical for the functional integrity of proteins because of theircapacity to form disulfide bonds and restrict secondary structure. Todetermine whether any of the cysteines in human chitinase are criticalfor enzymatic activity, each cysteine is mutated individually to aserine.

[0066] A 39 kDa C-terminally truncated fragment of the mature humanchitinase protein was prepared as described above in Examples 3 and 5 byintroduction of a stop codon after the codon for amino acid 373. This 39kDa fragment lacked seventy-two C-terminal amino acid residues of themature protein, including six cysteines, yet retained similar specificenzymatic activity compared to the full length recombinant humanchitinase. This result indicates that the missing seventy-two C-terminalresidues, including the six cysteines, are not required for enzymaticactivity.

[0067] Further C-terminal deletions may be prepared, e.g., by digestingthe 3′ end of the truncated human chitinase coding sequence described inExample 3 with exonuclease III for various amounts of time and thenligating the shortened coding sequence to plasmid DNA encoding stopcodons in all three reading frames. N-terminal deletions are prepared ina similar manner by digesting the 5′ end of the coding sequence and thenligating the digested fragments into a plasmid containing a promotersequence and an initiating methionine immediately upstream of thepromoter site. These N-terminal deletion analogs or fragments may alsobe expressed as fusion proteins.

[0068] Alternatively, human chitinase polypeptide analogs may also beprepared by full or partial chemical peptide synthesis using techniquesknown in the art. [See, e.g., synthesis of IL-8 in Clark-Lewis et al.,J. Biol Chem., 266:23128-34 (1991); synthesis of IL-3 in Clarke-Lewis etal., Science, 231:134-139 (1986); and synthesis by ligation in Dawson etal., Science, 266:776-779 (1994).] Such synthetic methods also allow theselective introduction of novel, unnatural amino acids and otherchemical modifications.

[0069] The biological activities, including enzymatic, anti-fungal, andextracellular matrix remodeling activities, of the human chitinasepolypeptide analogs are assayed by art-recognized techniques, such asthose described in Examples 9 to 15 below.

EXAMPLE 7 Production of Human Chitinase Chitin-binding Fragments andAnalogs Thereof

[0070] A. Generation of SEAP Fusion Proteins

[0071] The location of the chitin-binding domain of human chitinase wasdetermined by generating fusion proteins comprising N-terminallytruncated portions of human chitinase and testing these products forchitin-binding activity. First, a chimeric protein comprising fulllength secreted alkaline phosphatase (SEAP) protein (at the N-terminusof the chimeric protein) [Berger et al., Gene, 66:1-10 (1988)] fused tothe C-terminal 99 amino acids of human chitinase (at the C-terminus ofthe chimeric protein) was generated as follows. The SEAP component actsas a traceable marker of the chimeric protein.

[0072] The SEAP DNA was amplified from the pSEAP2-Control plasmid(Clontech, Palo Alto, Calif.) via polymerase chain reaction (PCR) withprimers SEAP Start (SEQ ID NO: 18) and SEAP Stop (SEQ ID NO: 19) thatintroduced a HindIII site to the 5′ end and a multiple cloning region tothe 3′ end. PCR was carried out using 100 ng of template DNA, 1 μg ofeach primer, 0.125 mM of each dNTP, 10 mM Tris-HCl, pH 8.4, 50 mM MgCl₂and 2.5 units of Taq polymerase, with an initial denaturation step of94° C. for four minutes followed by 30 cycles of amplication: 1 minuteat 94° C., 1 minute at 60° C., and 2 minutes at 72° C. ThisPCR-generated cDNA was cloned into the HindIII and ApaI sites of pcDNA3(Invitrogen, San Diego, Calif.) to generate a vector called pcDNA-SEAP.DNA encoding the C-terminal 99 amino acids of human chitinase (residues347-445) was also generated by PCR under the same conditions using theprimers indicated in Table 1 below, which introduced EcoRI and XbaIsites to the 5′ and 3′ ends. This PCR-generated chitinase DNA sequencewas cloned into the EcoRI and XbaI sites of the multiple cloning regionof pcDNA-SEAP.

[0073] The resulting construct encoding the chimera was transientlytransfected into COS 7 cells by incubation in Dulbecco's modified Eaglemedium (DMEM) containing 0.5 mg/ml DEAE dextran, 0.1 mM chloroquine and10 μg of plasmid DNA for 1.5 hours. The cells then were treated with 10%DMSO in phosphate buffered saline for 45 seconds, washed with serum-freemedium and incubated in DMEM supplemented with 1 mM L-glutamine, 100U/ml penicillin, 100 μg/ml streptomycin and 10% fetal calf serum. Afterfour days, the culture medium was assayed for SEAP activity as describedby Flanagan and Leder, Cell, 63:185-194 (1990). SEAP activity wasreadily detectable. Incubation of the culture medium containing thisfusion protein with insoluble chitin (Sigma, St. Louis, Mo.) for 1 hourat 4° C. resulted in precipitation of more than 80% of the SEAP activitywith the chitin. This result demonstrated that the entire chitin-bindingdomain is contained within the C-terminal 99 amino acids of humanchitinase.

[0074] DNA encoding additional chitin-binding domain truncates weregenerated by PCR and expressed as fusions with SEAP protein as describedabove. These fusion proteins were assayed for chitin binding activity,with results as displayed in Table 1 below. TABLE 1 Truncates Primersused Chitin-binding tested to generate truncates Activity Amino acidsSEAP CBD 1149 (SEQ ID NO: 20) and ⊕ 347-445 Hu Chit Stop (SEQ ID NO: 26)Amino acids SEAP CBD 1231 (SEQ ID NO: 21) and ⊕ 374-445 Hu Chit Stop(SEQ ID NO: 26) Amino acids SEAP CBD 1285 (SEQ ID NO: 22) and ⊕ 392-445Hu Chit Stop (SEQ ID NO: 26) Amino acids SEAP CBD 1309B (SEQ ID NO: 23)⊖ 400-445 and Hu Chit Stop (SEQ ID NO: 26) Amino acids SEAP CBD 1338(SEQ ID NO: 24) and ⊖ 409-445 Hu Chit Stop (SEQ ID NO: 26) Amino acidsSEAP CBD 1149 (SEQ ID NO: 20) and ⊖ 347-431 SEAP CBD 1357 (SEQ ID NO:25) Amino acids SEAP CBD 1231 (SEQ ID NO: 21) and ⊖ 374-431 SEAP CBD1357 (SEQ ID NO: 25) Amino acids SEAP CBD 1285 (SEQ ID NO: 22) and ⊖392-431 SEAP CBD 1357 (SEQ ID NO: 25) Amino acids SEAP CBD 1296 (SEQ IDNO: 35) and ⊕ 395-445 Hu Chit Stop (SEQ ID NO: 26) Amino acids SEAP CBD1300 (SEQ ID NO: 36) and ⊕ 397-445 Hu Chit Stop (SEQ ID NO: 26) Aminoacids SEAP CBD 1285 (SEQ ID NO: 22) and ⊖ 392-443 Hu Chit Stop 7 (SEQ IDNO: 37)

[0075] B. Generation of Cysteine Mutation Analogs

[0076] To determine whether any of the six cysteines within the 99C-terminal amino acids of human chitinase were critical for bindingchitin, analogs of chitinase fragments were prepared in which eachcysteine was mutated individually to a serine. Six PCR products in whicheach of the six cysteines was individually mutated to serine weregenerated using the primers indicated in Table 2 below and fused to SEAPcDNA as described above. Chimeric proteins produced by transientlytransfected COS cells were assayed for chitin-binding activity asdescribed above. The results of these experiments demonstrated that eachof the six cysteines is required for chitin-binding activity. TABLE 2Chitinase binding domain Primers used Chitin-binding analog tested togenerate analog activity C399S SEAP CBD dC1 (SEQ ID NO: 27) and ⊖ HuChit Stop (SEQ ID NO: 26) C419S SEAP CBD dC2 (SEQ ID NO: 28) and ⊖ HuChit Stop (SEQ ID NO: 26) C429S SEAP CBD 1285 (SEQ ID NO: 22) ⊖ and SEAPCBD dC3 (SEQ ID NO: 29) C439S SEAP CBD 1285 (SEQ ID NO: 22) ⊖ and SEAPCBD dC4 (SEQ ID NO: 30) C441S SEAP CBD 1285 (SEQ ID NO: 22) ⊖ and SEAPCBD dC5 (SEQ ID NO: 31) C442S SEAP CBD 1285 (SEQ ID NO: 22) ⊖ and SEAPCBD dC6 (SEQ ID NO: 32)

[0077] Additional chitin-binding fragments and fragment analogs thereofcan be prepared by recombinant techniques or by full or partial chemicalsynthesis as described in Example 6.

[0078] C. Expression of a Chitin-binding Fragment in Yeast

[0079] A chitin-binding domain fragment consisting of residues 392-445of SEQ ID NO: 2 was expressed at high levels in the yeast Saccharomycescerevisiae. An expression construct, α-FLAG-CBD, was designed in whichthe nucleotides corresponding residues 392-445 of SEQ ID NO: 2 werefused to the 3′ terminus of sequence encoding the S. cerevisiae α-factorpre-pro sequence [Brake et al., Proc. Natl. Acad. Sci. 81:4642-4646(1984)] and the FLAG epitope tag (Eastman Kodak). To accomplish this,PCR using primers CBDαFLAG (sense; SEQ ID NO: 33) and Hu Chit Stop 5(antisense; SEQ ID NO: 34) was conducted using full-length humanchitinase DNA as a template. The CBDαFLAG primer sequence contains anAsp 718 restriction endonuclease site upstream of a FLAG tag-encodingregion that is in-frame with the sequence that encodes the first eightamino acids of the chitin-binding domain fragment 392-445. The Hu ChitStop 5 primer sequence encodes the C-terminal seven amino acids of thechitin-binding domain fragment followed by Gly-Ala-Gly linked to sixhistidine residues (His₆) which precede a three amino acid segment priorto the translation termination codon. The His₆ tract is included tofacilitate purification of the expressed product by metal affinitychromatography [as described in Nilsson et al., Prot. Expr. Purification11:1-16 (1997)]. A Not I restriction endonuclease site was includedimmediately 3′ of the stop codon.

[0080] The PCR product generated with these primers was digested withAsp 718 and Not I and cloned into the corresponding sites within theexpression cassette of plasmid pAYE/VEC, which is derived by modifyingpAYE674 [Delta Biotechnology Limited; Sleep et al., Bio/technology9:183-187 (1991)] to add restriction sites to facilitate theincorporation of expression cassettes into pSAC/VEC (described below).The expression cassette within the resulting construct, designatedpAYE/AF/CBD, consisted of an in-frame fusion of the nucleotides encodingthe S. cerevisiae α-factor pre-prosequence and the chitin-binding domainfragment. Upon synthesis, the fusion protein is targeted to the membranewhere the mature FLAG-chitin-binding domain fragment-His₆ peptide isreleased from the α-factor pre-pro sequence by the action of the KEX2protease. Transcription of the α-factor pre-pro-CBD fusion is under thecontrol of the strong promoter PRB1 and the transcription terminationsequence from ADH1.

[0081] The expression cassette was excised from pAYE/AF/CBD by digestionwith Sfi I and Pac I and cloned into the corresponding sites ofpSAC/VEC, which is derived by modifying the disintegration vector pSAC35(Delta Biotechnology Limited; Sleep et al., supra) to incorporate amultiple cloning site. This shuttle vector pSAC35 comprises a complete 2micron plasmid with a LEU2d selectable marker and two repeated sequencesflanking the pUC-derived E. coli origin of replication and β-lactamaseresistance marker. The resulting pSAC2/AF/CBD plasmid was transformedgenerally according to Ito et al., J. Bacteriol. 153:163-168 (1983),into the S. cerevisiae host strain IE41 (a cir° leu2 pep4::URA3 L261;Sleep et al. supra) and selected by growth on leucine deficient media.Following the introduction of pSAC2/AF/CBD into the host strain therepeated sequences undergo a single crossover recombination event,eliminating the pUC sequence. This vector is autonomously replicated,highly stable and has been shown to secrete high levels of product whenits host is grown in either selective or non-selective media (Sleep etal., supra).

[0082] Following clonal selection, four of the yeast clones were grownfor 16 hrs in 2 ml of selective medium at 30° C. The cultures weresubsequently transferred to 18 ml of YEPD medium and grown an additional48 hrs at 30° C. The culture media were harvested and evaluated bySDS-PAGE for the presence of expressed recombinant protein. The gelshowed that recombinant chitin-binding domain fragment 392-445 wassecreted from all four clones but not from the empty vector control. Thesecreted protein was positively identified as a chitin-binding domainfragment by its reactivity with a chitin-binding domain-specificmonoclonal antibody, 243Q, produced as described in Example 8, on aWestern blot.

[0083] The secreted chitin-binding domain fragment is highly enriched inthe yeast media but is not pure. A preliminary, small-scale metalaffinity purification procedure was conducted to obtain pure material.Two ml of Chelating Sepharose Fast Flow (Pharmacia) was deposited into a12 ml drip column (BioRad). Ten ml of 50 mM NiSO₄ was applied to thechelating sepharose to charge it with nickel. Following a 10 ml washwith distilled water, the charged column was equilibrated with 10 ml ofBuffer A (20 mM Tris, pH 8, 0.5 M NaCl). Prior to loading, the pH of theculture medium from clone 34A was adjusted to 8 by addition of Tris pH 8to a final concentration of 20 mM. Fourteen ml of the medium was passedthrough the column, followed by a 10 ml wash with Buffer A. Recombinantprotein was subsequently eluted by sequential applications of Buffer Acontaining 10 (2ml), 50 (2 ml), or 100 mM (3 ml) imidazole. Ten μl ofeach fraction of the purification procedure was analyzed by SDS-PAGE.The gel showed that 100 mM imidazole eluted essentially pure recombinantchitin-binding domain fragment 392-445. Fractions 2 and 3 of the 100 mMimidazole elution were pooled; the pool was found to containapproximately 0.4 mg/ml of purified protein.

[0084] To assess functionality of the recombinant protein, 1 mg ofpowdered chitin was incubated with 25 μl of the purified chitin-bindingdomain fragment 392-445 for 1 hour at 4° C. Following the incubation,the insoluble chitin was removed by centrifugation and the amount ofprotein remaining in the supernatant was compared with that of achitin-free control medium by SDS-PAGE. An approximately 50% reductionof recombinant protein was observed in the medium treated with chitin.This demonstrates that a least a significant fraction of the recombinantprotein retains its capacity to bind chitin.

[0085] A second yeast expression construct was designed to express achitin-binding domain fragment consisting of residues 392-445 of SEQ IDNO: 2 without the FLAG epitope and His6 tags. The construct wasassembled as described above except that the PCR primers used to amplifythe chitin-binding domain-encoding region were CBDα (sense; SEQ ID NO:38) and Hu Chit Stop 4 (antisense; SEQ ID NO: 39). Cells weretransformed and selected as described above.

[0086] A transformed yeast clone expressing recombinant chitin-bindingdomain was grown overnight at 30° C. in 10 ml of SC-leu-ura medium(Bio101, Vista, Calif.) containing 2% glucose. One ml of this culturewas seeded into 100 ml of the same medium and incubated at 30° C. withshaking. After 19 hours, 65 ml of the culture was seeded into 1.2 L ofYB2V medium [0.2% glucose, 0.008% FeCl₃, 1 g/L Na₃ citrate, 25 g/Lcasamino acids, 13.5 g/L KH₂PO₄, 3.8 g/L (NH₄)₂SO₄, 4 mM MgSO₄, 0.0004%thiamine, trace metals and vitamins] in a 3 L fermentor. Thefermentation was carried out at 30° C., pH 5.5, with 1200 rpm agitationand an airflow rate of 3 L/min. The pH was controlled using phosphoricacid and ammonium hydroxide. The fermentor was operated under batchconditions for the first 13 hours, after which time feed addition wasinitiated. Feed medium YF6V.1 [50% glucose, 0.02% FeCl₃, 1 g/L Na₃citrate, 50 g/L casamino acids, 2.9 g/L KH₂PO₄, 5 g/L (NH₄)₂SO₄, 15.2 mMMgSO₄, 0.001% thiamine, trace metals and vitamins] was added to thefermentor at a constant rate of 3.6 ml/hour for 4 hours, then the feedrate was increased exponentially with a 5 hour doubling time to amaximum of 9.38 ml/hour. After a total fermentation time of 93 hours,cells were harvested by centrifugation. 1.6 L of the broth wascentrifuged for 40 minutes at 4° C., 5000×g. The cell pellet wasdiscarded and the supernatant was filtered through a pre-filter and a0.2 μM filter.

[0087] Purification of the recombinant chitin-binding domain wasaccomplished by passing culture medium from the fermentation through achitin bead column. A 25 ml bed volume of chitin beads (New EnglandBiolabs) was prepared in a 50 ml column (Amicon), prewashed with 250 mlof 1% SDS, and equilibrated with 250 ml Buffer A (20 mM Tris, pH 8, 500mM NaCl) at a flow rate of 2 ml/min. Clarified medium was passed throughthe chitin bead column at a rate of 1 ml/min. Following a 250 ml washwith Buffer A, protein was eluted from the beads with 50% acetonitrile,0.1% trifluoroactetate and collected into 4 ml fractions. Acetonitrilewas evaporated from the eluate by vacuum centrifugation. Analysis ofelution fractions by SDS-polyacrylamide gel electrophoresis and proteinquantitation revealed that 94% of the purified chitin-binding domain wasrecovered in 3 consecutive fractions. In total, 109 mg of chitin-bindingdomain was obtained from 130 ml of fermentation medium. Analysis of thepurified protein by MALDI mass spectrometry (Perseptive Biosystems)shows a single peak with molecular weight of 5911.9, a value thatcorresponds favorably to the predicted mass of 5909.8. In a test of itsfunctional integrity, 20 μg of the purified chitin-binding domain mixedwith 250 μl of chitin beads resulted in >95% of the peptide being boundto the beads.

EXAMPLE 8 Preparation of Monoclonal Antibodies to Human Chitinase

[0088] The following two protocols (multiple challenge or single shotimmunizations) may be used to generate monoclonal antibodies to humanchitinase. In the first protocol, a mouse is immunized by periodicinjection with recombinant human chitinase (e.g., 10-20 μg emulsified inFreund's Complete Adjuvant) obtained as described in any of Examples 3through 6. The mouse is given a final pre-fusion boost of humanchitinase in PBS, and four days later the mouse is sacrificed and itsspleen removed. The spleen is placed in 10 ml serum-free RPMI 1640, anda single cell suspension is formed by grinding the spleen between thefrosted ends of two glass microscope slides submerged in serum-free RPMI1640, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100units/ml penicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada).The cell suspension is filtered through sterile 70-mesh Nitex cellstrainer (Becton Dickinson, Parsippany, N.J.), and is washed twice bycentrifuging at 200 g for 5 minutes and resuspending the pellet in 20 mlserum-free RPMI. Splenocytes taken from three naive Balb/c mice areprepared in a similar manner and used as a control. NS-1 myeloma cells,kept in log phase in RPMI with 11% fetal bovine serum (FBS) (HycloneLaboratories, Inc., Logan, Utah) for three days prior to fusion, arecentrifuged at 200 g for 5 minutes, and the pellet is washed twice asdescribed in the foregoing paragraph.

[0089] One×10⁸ spleen cells are combined with 2.0×10⁷ NS-1 cells andcentrifuged, and the supernatant is aspirated. The cell pellet isdislodged by tapping the tube, and 1 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer Mannheim) is added with stirring over thecourse of 1 minute, followed by the addition of 7 ml of serum-free RPMIover 7 minutes. An additional 8 ml RPMI is added and the cells arecentrifuged at 200 g for 10 minutes. After discarding the supernatant,the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ splenocytes/ml andplated into 10 Corning flat-bottom 96-well tissue culture plates(Corning, Corning N.Y.).

[0090] On days 2, 4, and 6, after the fusion, 100 μl of medium isremoved from the wells of the fusion plates and replaced with freshmedium. On day 8, the fusion is screened by ELISA, testing for thepresence of mouse IgG binding to human chitinase as follows. Immulon 4plates (Dynatech, Cambridge, Mass.) are coated for 2 hours at 37° C.with 100 ng/well of human chitinase diluted in 25 mM Tris, pH 7.5. Thecoating solution is aspirated and 200 ul/well of blocking solution [0.5%fish skin gelatin (Sigma) diluted in CMF-PBS] is added and incubated for30 min. at 37° C. Plates are washed three times with PBS with 0.05%Tween 20 (PBST) and 50 μl culture supernatant is added. After incubationat 37° C. for 30 minutes, and washing as above, 50 μl of horseradishperoxidase conjugated goat anti-mouse IgG(fc) (Jackson ImmunoResearch,West Grove, Pa.) diluted 1:3500 in PBST is added. Plates are incubatedas above, washed four times with PBST, and 100 μL substrate, consistingof 1 mg/ml o-phenylene diamine (Sigma) and 0.1 μl/ml 30% H₂O₂ in 100 mMcitrate, pH 4.5, are added. The color reaction is stopped after 5minutes with the addition of 50 μl of 15% H₂SO₄. A₄₉₀ is read on a platereader (Dynatech). Selected fusion wells are cloned twice by dilutioninto 96-well plates and visual scoring of the number of colonies/wellafter 5 days. The monoclonal antibodies produced by hybridomas areisotyped using the Isostrip system (Boehringer Mannheim, Indianapolis,Ind.).

[0091] Alternatively, a second protocol utilizing a single-shotintrasplenic immunization may be conducted generally according to Spitz,Methods Enzymol., 121:33-41 (1986). The spleen of the animal is exposedand recombinant human chitinase (e.g., 10-20 μg in PBS at aconcentration of about 0.02% to 0.04%, with or without an aluminumadjuvant), obtained as described in any of Examples 3 through 6, isinjected, after which the spleen is returned to the peritoneal cavityand the animal is stitched closed. Three days later, the mouse issacrificed and its spleen removed. A spleen cell suspension is prepared,washed twice with RPMI 1640 supplemented with 3% fetal calf serum (FCS),and resuspended in 25 ml of the same medium. Myeloma cells (NS-O) arecollected at logarithmic growth phase, washed once and added to thespleen cell suspension in a 50 ml tube, at a ratio of 3:1 or 2:1 (spleencells:myeloma cells). The mixture is pelleted at about 450×g (1500 rpm),the supernatant aspirated, and the pellet loosened by tapping the tube.Fusion is performed at room temperature by adding 1 ml of polyethyleneglycol (PEG) 1500 over 1 minute, with constant stirring. The mixture isincubated for another minute, then 1 ml of warm RPMI (30 to 37° C.) isadded over 1 minute followed by 5 ml RPMI over 3 minutes and another 10ml RPMI over another 3 minutes. The cell suspension is centrifuged andresuspended in about 200 ml of HAT selective medium consisting of RPMI1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 20%FCS, 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine. Thecell suspension is dispensed in 1 ml volumes into tissue culture platesand incubated at 37° C. in a humid atmosphere with 5% CO₂-95% air for 8to 10 days. Supernatants are aspirated and the cells are fed with 1 mlHAT medium per well, every 2 to 3 days according to cell growth.Supernatants of confluent wells are screened for specific antibodies andpositive wells are cloned.

[0092] Using the above protocols, several monoclonal antibodies withreactivity to human chitinase were generated. For fusion 243, each offive 6-12 week old Balb/c mice was prebled on day 0 and then immunizedby subcutaneous injection with 10-20 μg recombinant human chitinaseprepared as described in Example 5, emulsified in complete Freundsadjuvant. On days 21, 42 and 60 each mouse was boosted with 50 μg of thesame recombinant human chitinase in incomplete Freunds adjuvant. Mouse#2483 was additionally given 20 μg of recombinant human chitinase dailyon days 216 through 219. On day 220 the spleen of mouse #2483 wasremoved sterilely and treated as described above. Briefly, a single-cellsuspension was formed by grinding the spleen between the frosted ends oftwo glass microscope slides submerged in serum free RPMI 1640,supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/mlpenicillin, and 100 μg/ml streptomycin (RPMI) (Gibco, Canada). The cellsuspension was filtered through a sterile cell strainer (BectonDickinson, Parsippany, N.J.), and washed twice with serum free RPMI bycentrifuging at 200×g for 5 minutes and resuspending the pellet in 20 mlserum free RPMI. Thymocytes taken from naive Balb/c mice were preparedin a similar manner.

[0093] NS-1 myeloma cells, kept in log phase in RPMI with 10% FetalClone serum (FCS) (Hyclone Laboratories, Logan, Utah) for three daysprior to fusion, were centrifuged at 200×g for 5 minutes, and the pelletwas washed twice and resuspended in 10 ml serum free RPMI as describedabove.

[0094] Spleen cells were combined with NS-1 cells at a ratio of 5:1,centrifuged at 200×g and the supernatant was aspirated. The cell pelletwas dislodged by tapping the tube and 2 ml of 37° C. PEG 1500 (50% in 75mM Hepes, pH 8.0) (Boehringer Mannheim) was added with stirring over thecourse of 1 minute, followed by adding 14 ml of serum free RPMI over 7minutes. An additional 16 ml RPMI was added and the cells werecentrifuged at 200×g for 10 minutes. After discarding the supernatant,the pellet was resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT; Gibco),25 units/ml recombinant human IL-6 (Boehringer Mannheim) and 1.5×10⁶thymocytes/ml. The suspension was dispensed into ten 96-well flat bottomtissue culture plates (Coring, United Kingdom) at 200 μl/well. Cells inplates were fed 3 to 5 times before screening by aspiratingapproximately 100 μl from each well with a 20-gauge needle (BectonDickinson), and adding 100 μl/well of the plating medium described aboveexcept containing 10 units/ml IL-6 and lacking thymocytes.

[0095] Supernatants from fusion 243 were screened initially by ELISA onthe immunogen (full length human chitinase), and detected with goatanti-mouse IgG (fc) horseradish peroxidase conjugate. To ensureclonality, positive wells chosen from each fusion were subcloned 4 timesby limiting dilution, using media lacking aminopterin. Cloning wascompleted for cell lines 243K, 243M and 243Q.

[0096] Isotypes were determined for the monoclonal antibodies from thetwo cell lines using either the Isostrip kit (Boehringer Mannheim) or anELISA using isotype specific reagents (Zymed Laboratories, South SanFrancisco, Calif.). All antibodies are IgG1 isotype.

[0097] To test whether any of these bear specificity toward thechitin-binding domain, each antibody was used to probe a western blotcontaining the full length chitinase, the C-terminally truncatedchitinase (amino acids 1-373), and the recombinant chitin-binding domain(amino acids 392-445) produced in yeast. Three antibodies, 243K, 243M,and 243Q, bound to the full-length chitinase and to the chitin-bindingdomain but not to the C-terminal truncate.

[0098] All combinations of all three antibodies were tested forapplicability to use in a sandwich ELISA format. Nunc-Immuno Moduleplates were coated with 125 μl of first antibody at 2 μg/ml andincubated overnight at 4° C. The antibody solution was then replacedwith 300 μl/well of blocking solution (5% Teleostean gelatin, 0.05%Proclin 300 in CMF-PBS). After a 30 min incubation at room temperature,the blocking solution was replaced with 20 ng/ml recombinantchitin-binding domain in Omni Diluent (1% Teleostean gelatin, 0.05%Tween 20, 0.05% Proclin in CMF-PBS) and incubated 30 min at 37° C. Wellswere washed 5 times with wash buffer (145 mM NaCl, 1.5% Tween 20) thenreceived 0.25 μg/ml of biotinylated second antibody. After a 30 minincubation at 37° C., wells were again washed 5 times, treated with 100μl of streptavidin-conjugated horseradish peroxidase (Pierce), andincubated 30 min at 37° C. Following another 5 washes, wells received100 μl of substrate (0.01 g/ml tetramethylbenzidine in dimethylsulfoxide diluted 1:100 into 100 mM sodium acetate trihydrate, pH 5.5,0.015% H₂O₂) and were incubated at room temperature in the dark. After30 min, 100 μl of 1N H₂SO₄ was added to stop the reaction and absorbanceat wavelengths of 450 and 630 nm was determined. This approachidentified 243Q (first antibody) and 243M (second antibody) as thecombination which delivered the greatest signal relative to all othercombinations. The hybridoma 243Q producing antibody 243Q was depositedon ______ (date) with American Type Culture Collection, 10801 UniversityBlvd., Manassas, Va. 20110-2209 and was assigned Accession No. ______.The hybridoma 243M producing antibody 243M was deposited on ______(date) with American Type Culture Collection, 10801 University Blvd.,Manassas, Va. 20110-2209 and was assigned Accession No. ______.

EXAMPLE 9 Catalytic Activity of Recombinant Chitinase

[0099] Chitotriosidase (chitinase) activity was measured using thefluorogenic substrate4-methylumbelliferyl-β-D-N,N′,N″-triacetylchitotriose (4MU-chitotrioside, Sigma Chemical, St. Louis, Mo.) in McIlvain buffer(Hollak et al., supra). Ten μl samples of the recombinant productdescribed in Example 5A were combined with 10 μl bovine serum albumin(10 mg/ml), 15 μl fluorogenic substrate (2.71 mM), and 65 μl buffer(0.1M citric acid, 0.2M sodium phosphate, pH 5.2) in a total volume of100 μl. Reactions were incubated at 37° C. for 15 minutes, then thereaction was stopped with the addition of 2 ml of 0.3M glycine/NaOHbuffer (pH 10.6). The fluorescent cleavage product,4-methylumbelliferone, was monitored with a fluorimeter (SLM-AMINCOInstruments, Inc., Rochester, N.Y.) at 450 nm. To obtain a standardcurve, several substrate concentrations were combined with excessbacterial chitinase to ensure that substrate was completely cleaved. Theknown quantity of 4-MU was then correlated to the fluorescence signalfrom the fluorimeter and linear regression was used to determine astandard curve. The signal produced with diluted purified recombinantchitinase in the assay was then used to interpolate the nmol quantity ofsubstrate cleaved by the enzyme during the reaction time. This numberwas then divided by the concentration of protein to obtain the nmol/minper mg protein (determined by A₂₈₀ and calculated molar extinctioncoefficient).

[0100] The chitotriosidase activity of the recombinant human chitinaseproduced in COS cells as described in Example SA was determined to be 90nmol/min per mg protein. Any of the human chitinase fragment products ofthe present invention can also be tested for chitinase enzymaticactivity in this manner.

EXAMPLE 10 Anti-fungal Activity of Chitinase Fragment Products in vitro

[0101] Conventional anti-fungal agents that have been conjugated tohuman chitinase products of the invention can be tested for inhibitionof fungal growth in vitro. The two fungi Candida albicans andAspergillus fumigatis are serious pathogens for immunocompromisedpatients. Both Candida and Aspergillus are grown in RPMI growth media at37° C. to approximately 10,000-50,000 colony forming units (CFU) per ml.Serial dilutions of the test drug are added to cultures, and fungalgrowth is assessed at 24 hours by turbidity of cultures.

[0102] The anti-fungal activity of the test drug may also be evaluatedin an agar diffusion assay, in a broth assay according to NationalCommittee on Clinical Laboratory Standards, and in a cell wallinhibition assay according to Selitrennikoff, Antimicrob. AgentsChemother., 23:757-765 (1983).

[0103] In the agar diffusion assay, approximately 1×10⁶ cells/ml ofCandida albicans (ATCC no. 90028) is inoculated into 1.5% agar (RPMI1640 media buffered with 2-(N-morpholino)propanesulfonic acid (MOPS), pH7.0. A disk containing a set amount, e.g., 50 μg of the test drug or acontrol is placed on the agar, and the zone of growth inhibition ismeasured.

[0104] In the broth assay, a set amount, e.g., 50 μg/ml of the test drugor a control is added with a certain concentration of the test fungalorganism to RPMI 1640 media buffered with MOPS, pH 7.0. The samples areincubated at 35° C., with shaking at 120 rpm, for 48 hours, and thengrowth is evaluated by measuring the turbidity of the suspension.Appropriate concentrations of test fungal organism include thefollowing: 2.5×10⁴ cells/ml of Candida albicans (ATCC no. 90028); 5×10⁴cells/ml of Candida albicans-polyene resistant (ATCC no. 38247); 1×10⁴cells/ml of Aspergillus fumigatus (ATCC no. 16424); 1×10⁴ cells/ml ofNeurospora crassa (ATCC no. 18889); and 1×10⁴ cells/ml of Saccharomycescerevisiae (ATCC no. 26108).

[0105] The os-1 whole cell assay, which identifies inhibitors of fungalcell wall biosynthesis, is conducted essentially according toSelitrennikoff, supra, using an inoculum of 1.5×10⁵ protoplasts/mlembedded in agar (Vogel's Medium N, 7.5% sorbitol, 1.5% sucrose, 10μg/ml nicotinamide and 1% agar) incubated at 25° C. for 72 hours. Thecultures are monitored for changes in growth and morphology after diskscontaining a set amount, e.g., 50 μg of test drug or control are placedon the agar medium. The os-1 cell is a mutant strain of Neurosporacrassa that grows as protoplasts without cell walls when incubated undercertain conditions at 37° C., but regenerates a cell wall under theappropriate conditions when the temperature is shifted to about 22° C.Samples that inhibit growth are considered fungal growth inhibitors andsamples that prevent cell wall regeneration, but do not kill the cells,are considered cell wall-specific inhibitors.

EXAMPLE 11 Anti-fungal Activity of Recombinant Chitinase in vivo in Mice

[0106] The pharmacokinetics of recombinant human chitinase in mice weredetermined as follows. Female Balb/c mice, 6-8 weeks old, wereadministered 0.5 mg/kg, 5.0 mg/kg and 50 mg/kg recombinant humanchitinase by intravenous injection in the tail vein. For each dose, micewere terminally bled at 0.01, 0.25, 1, 8 and 24 hours after injection (2animals were used per time point per dosage). Serum samples were thenassayed for chitinase activity and concentration. Results are shown inTable 3 below. TABLE 3 Dose AUC Vss cL MRT half-life Cmax (mg/kg)(μg/ml/h) (ml/kg) (ml/h/kg) (h) (h) (μg) 0.5 31.24 12.03 16.01 0.75 0.7422.30 5.0 278.50 13.61 17.95 0.76 1.38 162.84 50.0 2505.83 52.92 19.952.65 2.33 1179.19

[0107] The pharmacokinetics of chitinase fragment products of theinvention or therapeutic agents comprising such chitinase fragmentproducts may be assessed in the same manner.

[0108] Several animal models have been developed for testing efficacy ofanti-fungal compounds [see Louie et al., Infect. Immun., 62: 2761-2772,1994; Kinsman et al., Antimicrobial Agents and Chemotherapy, 37:1243-1246, 1993; Nakajima et al., Antimicrobial Agents and Chemotherapy39: 1517-1521, 1995; Tonetti et al., Eur. J. Immunol., 25:1559-1565(1995); Denning and Stevens, Antimicrob. Agents Chemother., 35:1329-1333(1991); see also Stevens, J. Mycol. Med., 6(suppl.I):7-10 (1996)].Briefly, the animal host is infected with the fungi, varying doses oftest drug are administered to the animals, and their survival ismeasured over time. Comparative experiments may be performed using aconventional anti-fungal agent alone or the same agent conjugated to achitinase fragment product, to determine if conjugation of the agent tothe chitin-binding fragment products improves its anti-fungal efficacy.Specifically, acute systemic candidiasis is achieved in mice byintraperitoneal or intravenous challenge of 10×10⁶ CFU Candida albicans.The therapeutic agents are administered before or at 1 to 5 hours afterchallenge, and the number of survivors is determined after five days. Inaddition, the mice can be sacrificed and fungal load can be determinedin specific organs such as brain, kidney, lung, liver and spleen.Alternatively, the mice are challenged with lower doses of fungi, e.g.,Aspergillus (8-10×10⁶ CFU) or Candida (1×10⁶ CFU), in which casesurvival can be measured at more distant time points, e.g., 45 days. Thelong term fungicidal/fungistatic activity of a test drug may beevaluated by continuing therapy for a week or more, e.g., 11 days, andfollowing the animals over several weeks, e.g., 18 days to one month.Effective anti-fungal agents enhance the long term survival of animalsand reduce fungal load in blood and organs.

EXAMPLE 12 Activity of Chitinase in vivo in a Rabbit Model of InvasiveAspergillosis

[0109] The efficacy of therapeutic agents comprising chitinase fragmentproducts is assessed in an immunosuppressed rabbit model of invasiveaspergillosis which has been used for over ten years to evaluate avariety of anti-fungal therapies. See, e.g., Andriole et al., Clin.Infect. Dis., 14(Suppl. 1):S134-S138 (1992). The study is conductedgenerally according to Patterson et al., Antimicrob. Agents Chemother.,37.2307-2310 (1993) or George et al., J. Infect. Dis., 168:692-698(1993). Briefly, on day one the rabbits are given cyclophosphamide (200mg) intravenously to render them leukopenic, followed by triamcinoloneacetonide (10 mg) subcutaneously each day for the duration of theexperiment. On day two, 24 hours after immunosuppression, the animalsare challenged intravenously with about 10⁶ (lethal challenge) or about10⁵ (sublethal challenge) A. fumigatus conidia. Anti-fungal therapy withthe test agents is initiated at 24 hours after challenge or 48 hoursbefore challenge (for prophylaxis) and is continued for 5 to 6 days oruntil death. Exemplary doses of conventional anti-fungal agents are 1.5or 0.5 mg/kg/day intravenous amphotericin B, 60 or 120 mg/kg/day oralfluconazole and 100 mg/kg/day oral 5-fluorocytosine. Control rabbits arenot treated with any anti-fungal agent.

[0110] At autopsy or death, semiquantitative fungal cultures andhistopathologic examination are conducted on the liver, spleen, kidneys,lungs and brain. Cultures of the heart, urine and blood may also beperformed. Blood samples are obtained at intervals and assayed for whiteblood cell counts and circulating Aspergillus carbohydrate antigen usingan ELISA assay. The effect of treatment with the test drug is evaluatedon three endpoints: reduction in mortality rate, reduction in number ofAspergillus organisms cultured from target organs (fungal burden), andreduction in level of circulating Aspergillus antigen. Effectiveanti-fungal agents reduce mortality and/or fungal load.

[0111] Alternatively, pulmonary aspergillosis may be evaluated in thismodel generally according to Chilvers et al., Mycopathologia, 108:163-71(1989), in which the immunosuppressed rabbits are challenged withintratracheal instillation of Aspergillus fumigatus conidia, followed bybronchoalveolar lavage on days 1, 2, 4, 7 and 10 following challenge;fungal culture, chitin assay, white cell counts and histopathology areperformed on the lavage fluids to determine infective load within thelung. Effective anti-fungal agents reduce the infective load orinflammation within the lung.

EXAMPLE 13 Activity of Chitinase in vivo in a Rabbit Model ofDisseminated Candidiasis

[0112] The efficacy of therapeutic agents comprising chitinase fragmentproducts is assessed in a rabbit model of disseminated candidiasisgenerally according to Rouse et al., Antimicrob. Agents Chemother.,36:56-58 (1992). New Zealand white rabbits are infected systemicallywith about 3×10⁶ Candida albicans blastospores. Anti-fungal therapy withthe test drugs is initiated 48 hours after challenge with Candida (orbefore challenge for prophylaxis) and is continued for, e.g., four days.Surviving animals are sacrificed, and fungal cultures are performed onthe aortic valve with attached vegetation, lung, kidney and spleen.Fungal cultures and histopathological examination may also be performedon these and other organs, such as liver, brain, and heart. Urine andblood cultures may also be done. The effect of the anti-fungal therapyon mortality and circulating or tissue fungal burden is determined.

[0113] Bayer et al., Antimicrob. Agents Chemother., 19:179-184 (1981),describes a model in which rabbits are inoculated intraperitoneally withabout 5×10⁸ CFU Candida albicans. A saline peritoneal aspirate isobtained and cultured from each animal four days after intraperitonealinoculation, and animals with a positive fungal culture aspirate arerandomly assigned to control or treatment groups. Anti-fungal treatmentwith the test agents is begun seven days after challenge. The eyes ofall rabbits are evaluated using indirect ophthalmoscopy, as disseminatedcandidiasis may result in Candida endophthalmitis. Animals aresacrificed at 7, 11 and 14 days after initiation of therapy and theirabdomens inspected for evidence of peritonitis and intraabdominalabscess formation. Eyes are examined for macroscopic lesions. Tissuesamples from peritoneal abscesses, all other visible abscesses, kidneys,livers, spleens and ocular structures are weighed, homogenized in brainheart infusion broth, serially diluted and cultured to determine the CFUper gram of tissue. Renal and peritoneal abscesses are also fixed in 10%neutral formaldehyde and examined for histopathology. Sections arestained with periodic acid-Schiff reagent to determine the fungal burdenand fungal morphology. Effect of the test drugs on improving survivaland reducing fungal burden is evaluated.

EXAMPLE 14 Activity of Chitinase in vivo in a Rabbit Model of FungalEndophthalmitis

[0114] The efficacy of therapeutic agents comprising chitinase fragmentproducts is assessed in a rabbit model of Candida endophthalmitis,generally according to Park et al., Antimicrob. Agents Chemother.,39:958-963 (1995). Briefly, New Zealand albino rabbits, 2 to 2.5 kg, areinfected with an intravitreal inoculation of about 1,000 CFU of Candidaalbicans. Endophthalmitis is confirmed 5 days after inoculation byindirect ophthalmoscopy, and is defined as moderate to severe vitreoushaze with partial or complete obscuration of greater than 50% of theretinal and choroidal vasculature. The vitreous turbidity is graded on ascale, and the fundus appearance may be graded and documented by fundusphotography. The rabbits are then treated with test agents for 2 to 4weeks. Exemplary doses of conventional anti-fungal agents are 80mg/kg/day of oral fluconazole and 100 mg/kg every 12 hours of oral5-fluorocytosine.

[0115] The treatment effect is assessed at 2 and 4 weeks after therapyby indirect ophthalmoscopy, quantitative fungal culture, andhistopathology. For quantitative fungal culture, the eyes are dissectedand weighed, and a weighed fraction of each sample is homogenized andcultured on brucella agar-5% horse blood plates for 48 hours at 35° C.in 5 to 10% CO₂. The homogenized sample may also be diluted 10- or100-fold with sterile saline before plating. The colonies are countedand the total CFU in the eye calculated on the basis of the growthyielded from the measured fractions of sample. Treatment effect isassessed in terms of a reduction in the total intraocular fungal burden.For histopathology, representative eyes are removed, fixed in formalin,embedded in plastic, and sliced into 5 μm sections. The sections arestained with hematoxylin-eosin or Gomori's methenamine silver stain andexamined by light microscopy for inflammation, fibrous organization andfungal elements. The effect of the anti-fungal agents on reducingmortality, reducing fungal load, or reducing the inflammation associatedwith fungal infection, is evaluated.

[0116] Alternatively, a rabbit model of Aspergillus endophthalmitis maybe used generally according to Jain et al., Doc. Ophthalmol.,69:227-235(1988). Briefly, New Zealand white rabbits are inoculated in one eyewith about forty spores of Aspergillus fumigatus. Their contralateral(control) eyes receive a similar but sterile inoculum. After treatmentwith the test agents, the rabbits' eyes may be evaluated for clinicalappearance, electroretinogram waveforms, indirect ophthalmoscopy,quantitative fungal culture, and histopathology. Clinically evidentendophthalmitis typically develops within three to seven days afterinoculation.

EXAMPLE 15 Activity of Chitinase in vivo in a Rabbit Model of FungalEndocarditis

[0117] The efficacy of therapeutic agents comprising chitinase fragmentproducts is assessed in a rabbit model of Candida endocarditis generallyaccording to Witt and Bayer, Antimicrob. Agents Chemother., 35:2481-2485(1991). See also Longman et al., Rev. Infect. Dis., 12(Suppl.3):S294-298 (1990). Sterile thrombotic endocarditis is produced in NewZealand white rabbits by transaortic valvular placement of a sterilepolyethylene catheter (internal diameter, 0.86 mm), which remained inplace for the duration of the study. Infective endocarditis is thenestablished 48 hours after catheterization by intravenous injection ofabout 2×10⁷ C. albicans blastospores. Alternatively, C. parapsilosis maybe used. Anti-fungal therapy with test agents is initiated either 24hours before or 24 to 60 hours after fungal challenge. Therapy iscontinued daily for 9 or 12 days. Exemplary doses of conventionalanti-fungal agents are 1 mg/kg/day intravenous amphotericin B, 50mg/kg/day or 100 mg/kg/day intravenous or intraperitoneal fluconazole.Control rabbits are given no anti-fungal agent. At sacrifice, hearts areremoved and the position of the indwelling catheter verified. Cardiacvegetations from each animal are removed, pooled, weighed andhomogenized in 1 ml of sterile saline. The homogenate is seriallydiluted and quantitatively cultured on yeast potassium dextrose agar at35° C. for 48 hours. Culture-negative vegetations are considered tocontain less than 2 log₁₀ CFU/gram on the basis of average vegetationweight.

EXAMPLE 16 Chitin-binding and Chitin Hydrolytic Activity of Fragments ofHuman Chitinase

[0118] A. The Chitin-binding Domain is Essential for Binding ofChitinase to Chitin

[0119] The chitin-binding and chitotriosidase activity of full lengthchitinase (445 amino acids) was compared to the activity of theC-terminally truncated fragment (amino acids 1-373) described in Example5 above. Full length chitinase and chitinase fragment were prepared asfollows. The expression constructs for full-length chitinase (MO-13B)and for the 373 amino acid C-terminally truncated fragment, describedabove in Example 5, were transfected into COS cells. After 24 hours,culture media [Dulbecco's modified Eagle medium (Gibco)+10% fetal bovineserum+1 mM L-glutamine+100 U/ml penicillin+100 μg/ml streptomycin] werereplaced with media lacking fetal bovine serum. Cells were cultured foranother 3 days, after which media were harvested and assayed forhydrolytic and chitin-binding activities.

[0120] Levels of chitotriosidase activity, determined as described inExample 9, were nearly identical in culture media obtained from cellsexpressing full-length chitinase (57.6 nmol/ml/min) and culture mediafrom cells expressing the C-terminally truncated fragment (57.8nmol/ml/min). This is consistent with results reported in Example 6. Thesubstrate used for determining chitotriosidase activity (as in Example9) is triacetylchitotriose, a soluble, three residue oligosaccharide.

[0121] To compare chitin-binding activity, crab shell chitin (Sigma) wasground to a fme powder using a mortar and pestle, washed three timeswith equilibration buffer (20 mM Tris, pH 8, 500 mM NaCl), andresuspended to 100 mg/ml. One hundred μl of the chitin suspension wasadded to 1 ml of transfected COS cell medium and the mixture wasincubated 4 hr at 4° C. with continuous end-over-end mixing. Followingincubation, the chitin was pelleted by centrifugation (5 min, 12,000×g).Equivalent volumes of supernatant were supplemented with Laemmli bufferand 50 mM dithiothreitol (DTT), boiled, and electrophoresed through a12% polyacrylamide gel (Novex). Subsequent analysis of the gel byWestern blotting using a chitinase-specific monoclonal antibody (206A)revealed that no full length chitinase remained in the supernatant,i.e., that all of the full length chitinase had bound to the chitin andhad been pelleted with the chitin. In contrast, there was no discerniblereduction in the quantity of C-terminally truncated fragment (aminoacids 1-373) in the supernatant, indicating that the truncate had notbound to the chitin.

[0122] These observations indicate that the C-terminal 72 amino acids ofhuman chitinase are required for chitin-binding activity but not forhydrolysis of triacetylchitotriose.

[0123] B. The Chitin-binding Domain is Essential for Hydrolysis ofChitin But Not Triacetylchitotriose

[0124] The substrate used in Example 16A above for determiningchitotriosidase activity is a soluble, three residue oligosaccharide.Native chitin, however, is a long chain, insoluble polysaccharide. Thus,it is possible that the ability of an enzyme to hydrolyze the smallanalog may not predict its ability to hydrolyze chitin. To compare thechitinolytic activity of full-length chitinase (445 amino acids) and theC-terminally truncated fragment (amino acids 1-373), crab shell chitinwas incorporated into an agarose gel. Wells were cut into the solidifiedgel and loaded with either the full length chitinase or the truncate.After incubation, zones of clearing around the well indicated the extentof hydrolysis of the chitin.

[0125] The experiment was conducted as follows. A suspension of 0.4%chitin and 1.5% agarose was boiled in 20 mM sodium phosphate, pH 6,poured to a thickness 5 of 2 mm in a 10 cm petri dish, and allowed tosolidify. Wells 3 mm in diameter were cut into the agarose/chitinmatrix. Prior to loading wells, the recombinant proteins in the mediafrom transfected COS cells were concentrated 70-fold over a 30,000molecular weight cutoff filter device (Millipore). Equivalent quantitiesof the full-length and C-terminally truncated recombinant proteins wereloaded into adjacent wells. A third well was loaded with concentratedmedia from mock-transfected COS cells. Repeated equivalent loadings ofeach well were required in order to produce a visible zone of clearing.A zone of clearing that extended 3 mm from the periphery of the well wasobserved surrounding the well loaded with concentrated medium from COScells producing full-length chitinase. No clearing was seen around thewells containing either the truncate-containing media or mocktransfected cell culture media.

[0126] These observations demonstrate that the C-terminal 72 amino acidsof human chitinase are required for hydrolysis of chitin.

EXAMPLE 17 Chemical Modification of Recombinant Chitin-binding Domain byConjugation to Other Agents

[0127] In order to test whether the chitin-binding domain is amenable toserving as a carrier for small molecule pharmaceuticals, achitin-binding domain consisting of amino acids 392-445 of humanchitinase was chemically conjugated with either biotin or rhodamine, asfollows.

[0128] Biotinylation was accomplished using the Pierce EZ-linkSulfo-NHS-Biotinylation Kit (Pierce) according to the manufacturer'sprotocol. The NHS linkage was expected to target free amines which, onthe chitin-binding domain, are found at the N-terminus and at lysines402 and 440. Consistent with these expectations, MALDI mass spectrometryrevealed three biotinylated species with masses corresponding to thepresence of one, two, or three biotin molecules per peptide. Themajority (>60%) was triple biotinylated.

[0129] Using a succinimidyl linkage (Molecular Probes), rhodamine wasattached to the N-terminus of the chitin-binding domain.

[0130] Binding of either the biotin- or rhodamine-labeled peptide tochitin was indistinguishable from the binding properties of thenon-labeled peptide. This suggests that the chitin-binding domain isable to tolerate some chemical modification without an impact on itschitin-binding activity.

[0131] Numerous modifications and variations of the above-describedinvention are expected to occur to those of skill in the art.Accordingly, only such limitations as appear in the appended claimsshould be placed thereon.

1 39 1 1636 DNA Homo sapiens CDS (2)..(1399) mat_peptide (65)..(1399) 1c atg gtg cgg tct gtg gcc tgg gca ggt ttc atg gtc ctg ctg atg atc 49 MetVal Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met Ile -20 -15 -10cca tgg ggc tct gct gca aaa ctg gtc tgc tac ttc acc aac tgg gcc 97 ProTrp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala -5 -1 1 5 10cag tac aga cag ggg gag gct cgc ttc ctg ccc aag gac ttg gac ccc 145 GlnTyr Arg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro 15 20 25 agcctt tgc acc cac ctc atc tac gcc ttc gct ggc atg acc aac cac 193 Ser LeuCys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His 30 35 40 cag ctgagc acc act gag tgg aat gac gag act ctc tac cag gag ttc 241 Gln Leu SerThr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe 45 50 55 aat ggc ctgaag aag atg aat ccc aag ctg aag acc ctg tta gcc atc 289 Asn Gly Leu LysLys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile 60 65 70 75 gga ggc tggaat ttc ggc act cag aag ttc aca gat atg gta gcc acg 337 Gly Gly Trp AsnPhe Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr 80 85 90 gcc aac aac cgtcag acc ttt gtc aac tcg gcc atc agg ttt ctg cgc 385 Ala Asn Asn Arg GlnThr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg 95 100 105 aaa tac agc tttgac ggc ctt gac ctt gac tgg gag tac cca gga agc 433 Lys Tyr Ser Phe AspGly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser 110 115 120 cag ggg agc cctgcc gta gac aag gag cgc ttc aca acc ctg gta cag 481 Gln Gly Ser Pro AlaVal Asp Lys Glu Arg Phe Thr Thr Leu Val Gln 125 130 135 gac ttg gcc aatgcc ttc cag cag gaa gcc cag acc tca ggg aag gaa 529 Asp Leu Ala Asn AlaPhe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu 140 145 150 155 cgc ctt cttctg agt gca gcg gtt cca gct ggg cag acc tat gtg gat 577 Arg Leu Leu LeuSer Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp 160 165 170 gct gga tacgag gtg gac aaa atc gcc cag aac ctg gat ttt gtc aac 625 Ala Gly Tyr GluVal Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn 175 180 185 ctt atg gcctac gac ttc cat ggc tct tgg gag aag gtc acg gga cat 673 Leu Met Ala TyrAsp Phe His Gly Ser Trp Glu Lys Val Thr Gly His 190 195 200 aac agc cccctc tac aag agg caa gaa gag agt ggt gca gca gcc agc 721 Asn Ser Pro LeuTyr Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser 205 210 215 ctc aac gtggat gct gct gtg caa cag tgg ctg cag aag ggg acc cct 769 Leu Asn Val AspAla Ala Val Gln Gln Trp Leu Gln Lys Gly Thr Pro 220 225 230 235 gcc agcaag ctg atc ctt ggc atg cct acc tac gga cgc tcc ttc aca 817 Ala Ser LysLeu Ile Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr 240 245 250 ctg gcctcc tca tca gac acc aga gtg ggg gcc cca gcc aca ggg tct 865 Leu Ala SerSer Ser Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser 255 260 265 ggc actcca ggc ccc ttc acc aag gaa gga ggg atg ctg gcc tac tat 913 Gly Thr ProGly Pro Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr 270 275 280 gaa gtctgc tcc tgg aag ggg gcc acc aaa cag aga atc cag gat cag 961 Glu Val CysSer Trp Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln 285 290 295 aag gtgccc tac atc ttc cgg gac aac cag tgg gtg ggc ttt gat gat 1009 Lys Val ProTyr Ile Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp 300 305 310 315 gtggag agc ttc aaa acc aag gtc agc tat ctg aag cag aag gga ctg 1057 Val GluSer Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu 320 325 330 ggcggg gcc atg gtc tgg gca ctg gac tta gat gac ttt gcc ggc ttc 1105 Gly GlyAla Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe 335 340 345 tcctgc aac cag ggc cga tac ccc ctc atc cag acg cta cgg cag gaa 1153 Ser CysAsn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu 350 355 360 ctgagt ctt cca tac ttg cct tca ggc acc cca gag ctt gaa gtt cca 1201 Leu SerLeu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val Pro 365 370 375 aaacca ggt cag ccc tct gaa cct gag cat ggc ccc agc cct gga caa 1249 Lys ProGly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly Gln 380 385 390 395gac acg ttc tgc cag ggc aaa gct gat ggg ctc tat ccc aat cct cgg 1297 AspThr Phe Cys Gln Gly Lys Ala Asp Gly Leu Tyr Pro Asn Pro Arg 400 405 410gaa cgg tcc agc ttc tac agc tgt gca gcg ggg cgg ctg ttc cag caa 1345 GluArg Ser Ser Phe Tyr Ser Cys Ala Ala Gly Arg Leu Phe Gln Gln 415 420 425agc tgc ccg aca ggc ctg gtg ttc agc aac tcc tgc aaa tgc tgc acc 1393 SerCys Pro Thr Gly Leu Val Phe Ser Asn Ser Cys Lys Cys Cys Thr 430 435 440tgg aat tgagtcgcta aagcccctcc agtcccagct ttgaggctgg gcccaggatc 1449 TrpAsn 445 actctacagc ctgcctcctg ggttttccct gggggccgca atctggctcctgcaggcctt 1509 tctgtggtct tcctttatcc aggctttctg ctctcagcct tgccttccttttttctgggt 1569 ctcctgggct gcccctttca cttgcaaaat aaatctttgg tttgtgcccctcttcccaaa 1629 aaaaaaa 1636 2 466 PRT Homo sapiens 2 Met Val Arg SerVal Ala Trp Ala Gly Phe Met Val Leu Leu Met Ile -20 -15 -10 Pro Trp GlySer Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala -5 -1 1 5 10 Gln TyrArg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro 15 20 25 Ser LeuCys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His 30 35 40 Gln LeuSer Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe 45 50 55 Asn GlyLeu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile 60 65 70 75 GlyGly Trp Asn Phe Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr 80 85 90 AlaAsn Asn Arg Gln Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg 95 100 105Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser 110 115120 Gln Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln 125130 135 Asp Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu140 145 150 155 Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr TyrVal Asp 160 165 170 Ala Gly Tyr Glu Val Asp Lys Ile Ala Gln Asn Leu AspPhe Val Asn 175 180 185 Leu Met Ala Tyr Asp Phe His Gly Ser Trp Glu LysVal Thr Gly His 190 195 200 Asn Ser Pro Leu Tyr Lys Arg Gln Glu Glu SerGly Ala Ala Ala Ser 205 210 215 Leu Asn Val Asp Ala Ala Val Gln Gln TrpLeu Gln Lys Gly Thr Pro 220 225 230 235 Ala Ser Lys Leu Ile Leu Gly MetPro Thr Tyr Gly Arg Ser Phe Thr 240 245 250 Leu Ala Ser Ser Ser Asp ThrArg Val Gly Ala Pro Ala Thr Gly Ser 255 260 265 Gly Thr Pro Gly Pro PheThr Lys Glu Gly Gly Met Leu Ala Tyr Tyr 270 275 280 Glu Val Cys Ser TrpLys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln 285 290 295 Lys Val Pro TyrIle Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp 300 305 310 315 Val GluSer Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu 320 325 330 GlyGly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe 335 340 345Ser Cys Asn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu 350 355360 Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val Pro 365370 375 Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly Gln380 385 390 395 Asp Thr Phe Cys Gln Gly Lys Ala Asp Gly Leu Tyr Pro AsnPro Arg 400 405 410 Glu Arg Ser Ser Phe Tyr Ser Cys Ala Ala Gly Arg LeuPhe Gln Gln 415 420 425 Ser Cys Pro Thr Gly Leu Val Phe Ser Asn Ser CysLys Cys Cys Thr 430 435 440 Trp Asn 445 3 1656 DNA Homo sapiens CDS(27)..(1424) mat_peptide (90)..(1424) 3 gctgcagcct gccgctgagc tgcatc atggtg cgg tct gtg gcc tgg gca ggt 53 Met Val Arg Ser Val Ala Trp Ala Gly-20 -15 ttc atg gtc ctg ctg atg atc cca tgg ggc tct gct gca aaa ctg gtc101 Phe Met Val Leu Leu Met Ile Pro Trp Gly Ser Ala Ala Lys Leu Val -10-5 -1 1 tgc tac ttc acc aac tgg gcc cag tac aga cag ggg gag gct cgc ttc149 Cys Tyr Phe Thr Asn Trp Ala Gln Tyr Arg Gln Gly Glu Ala Arg Phe 5 1015 20 ctg ccc aag gac ttg gac ccc agc ctt tgc acc cac ctc atc tac gcc197 Leu Pro Lys Asp Leu Asp Pro Ser Leu Cys Thr His Leu Ile Tyr Ala 2530 35 ttc gct ggc atg acc aac cac cag ctg agc acc act gag tgg aat gac245 Phe Ala Gly Met Thr Asn His Gln Leu Ser Thr Thr Glu Trp Asn Asp 4045 50 gag act ctc tac cag gag ttc aat ggc ctg aag aag atg aat ccc aag293 Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys Met Asn Pro Lys 5560 65 ctg aag acc ctg tta gcc atc gga ggc tgg aat ttc agc act cag aag341 Leu Lys Thr Leu Leu Ala Ile Gly Gly Trp Asn Phe Ser Thr Gln Lys 7075 80 ttc aca gat atg gta gcc acg gcc aac aac cgt cag acc ttt gtc aac389 Phe Thr Asp Met Val Ala Thr Ala Asn Asn Arg Gln Thr Phe Val Asn 8590 95 100 tcg gcc atc agg ttt ctg cgc aaa tac agc ttt gac ggc ctt gacctt 437 Ser Ala Ile Arg Phe Leu Arg Lys Tyr Ser Phe Asp Gly Leu Asp Leu105 110 115 gac tgg gag tac cca gga agc cag ggg agc cct gcc gta gac aaggag 485 Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Ala Val Asp Lys Glu120 125 130 cgc ttc aca acc ctg gta cag gac ttg gcc aat gcc ttc cag caggaa 533 Arg Phe Thr Thr Leu Val Gln Asp Leu Ala Asn Ala Phe Gln Gln Glu135 140 145 gcc cag acc tca ggg aag gaa cgc ctt ctt ctg agt gca gcg gttcca 581 Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser Ala Ala Val Pro150 155 160 gct ggg cag acc tat gtg gat gct gga tac gag gtg gac aaa atcgcc 629 Ala Gly Gln Thr Tyr Val Asp Ala Gly Tyr Glu Val Asp Lys Ile Ala165 170 175 180 cag aac ctg gat ttt gtc aac ctt atg gcc tac gac ttc catggc tct 677 Gln Asn Leu Asp Phe Val Asn Leu Met Ala Tyr Asp Phe His GlySer 185 190 195 tgg gag aag gtc acg gga cat aac agc ccc ctc tac aag aggcaa gaa 725 Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr Lys Arg GlnGlu 200 205 210 gag agt ggt gca gca gcc agc ctc aac gtg gat gct gct gtgcaa cag 773 Glu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp Ala Ala Val GlnGln 215 220 225 tgg ctg cag aag ggg acc cct gcc agc aag ctg atc ctt ggcatg cct 821 Trp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile Leu Gly MetPro 230 235 240 acc tac gga cgc tcc ttc aca ctg gcc tcc tca tca gac accaga gtg 869 Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser Asp Thr ArgVal 245 250 255 260 ggg gcc cca gcc aca ggg tct ggc act cca ggc ccc ttcacc aag gaa 917 Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro Phe ThrLys Glu 265 270 275 gga ggg atg ctg gcc tac tat gaa gtc tgc tcc tgg aagggg gcc acc 965 Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp Lys GlyAla Thr 280 285 290 aaa cag aga atc cag gat cag aag gtg ccc tac atc ttccgg gac aac 1013 Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile Phe ArgAsp Asn 295 300 305 cag tgg gtg ggc ttt gat gat gtg gag agc ttc aaa accaag gtc agc 1061 Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys Thr LysVal Ser 310 315 320 tat ctg aag cag aag gga ctg ggc ggg gcc atg gtc tgggca ctg gac 1109 Tyr Leu Lys Gln Lys Gly Leu Gly Gly Ala Met Val Trp AlaLeu Asp 325 330 335 340 tta gat gac ttt gcc ggc ttc tcc tgc aac cag ggccga tac ccc ctc 1157 Leu Asp Asp Phe Ala Gly Phe Ser Cys Asn Gln Gly ArgTyr Pro Leu 345 350 355 atc cag acg cta cgg cag gaa ctg agt ctt cca tacttg cct tca ggc 1205 Ile Gln Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr LeuPro Ser Gly 360 365 370 acc cca gag ctt gaa gtt cca aaa cca ggt cag ccctct gaa cct gag 1253 Thr Pro Glu Leu Glu Val Pro Lys Pro Gly Gln Pro SerGlu Pro Glu 375 380 385 cat ggc ccc agc cct gga caa gac acg ttc tgc cagggc aaa gct gat 1301 His Gly Pro Ser Pro Gly Gln Asp Thr Phe Cys Gln GlyLys Ala Asp 390 395 400 ggg ctc tat ccc aat cct cgg gaa cgg tcc agc ttctac agc tgt gca 1349 Gly Leu Tyr Pro Asn Pro Arg Glu Arg Ser Ser Phe TyrSer Cys Ala 405 410 415 420 gcg ggg cgg ctg ttc cag caa agc tgc ccg acaggc ctg gtg ttc agc 1397 Ala Gly Arg Leu Phe Gln Gln Ser Cys Pro Thr GlyLeu Val Phe Ser 425 430 435 aac tcc tgc aaa tgc tgc acc tgg aattgagtcgcta aagcccctcc 1444 Asn Ser Cys Lys Cys Cys Thr Trp Asn 440 445agtcccagct ttgaggctgg gcccaggatc actctacagc ctgcctcctg ggttttccct 1504gggggccgca atctggctcc tgcaggcctt tctgtggtct tcctttatcc aggctttctg 1564ctctcagcct tgccttcctt ttttctgggt ctcctgggct gcccctttca cttgcaaaat 1624aaatctttgg tttgtgcccc tcaaaaaaaa aa 1656 4 466 PRT Homo sapiens 4 MetVal Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met Ile -20 -15 -10Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala -5 -1 15 10 Gln Tyr Arg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro 1520 25 Ser Leu Cys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His 3035 40 Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe 4550 55 Asn Gly Leu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile 6065 70 75 Gly Gly Trp Asn Phe Ser Thr Gln Lys Phe Thr Asp Met Val Ala Thr80 85 90 Ala Asn Asn Arg Gln Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg95 100 105 Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro GlySer 110 115 120 Gln Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr LeuVal Gln 125 130 135 Asp Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr SerGly Lys Glu 140 145 150 155 Arg Leu Leu Leu Ser Ala Ala Val Pro Ala GlyGln Thr Tyr Val Asp 160 165 170 Ala Gly Tyr Glu Val Asp Lys Ile Ala GlnAsn Leu Asp Phe Val Asn 175 180 185 Leu Met Ala Tyr Asp Phe His Gly SerTrp Glu Lys Val Thr Gly His 190 195 200 Asn Ser Pro Leu Tyr Lys Arg GlnGlu Glu Ser Gly Ala Ala Ala Ser 205 210 215 Leu Asn Val Asp Ala Ala ValGln Gln Trp Leu Gln Lys Gly Thr Pro 220 225 230 235 Ala Ser Lys Leu IleLeu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr 240 245 250 Leu Ala Ser SerSer Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser 255 260 265 Gly Thr ProGly Pro Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr 270 275 280 Glu ValCys Ser Trp Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln 285 290 295 LysVal Pro Tyr Ile Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp 300 305 310315 Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu 320325 330 Gly Gly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe335 340 345 Ser Cys Asn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg GlnGlu 350 355 360 Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu GluVal Pro 365 370 375 Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro SerPro Gly Gln 380 385 390 395 Asp Thr Phe Cys Gln Gly Lys Ala Asp Gly LeuTyr Pro Asn Pro Arg 400 405 410 Glu Arg Ser Ser Phe Tyr Ser Cys Ala AlaGly Arg Leu Phe Gln Gln 415 420 425 Ser Cys Pro Thr Gly Leu Val Phe SerAsn Ser Cys Lys Cys Cys Thr 430 435 440 Trp Asn 445 5 18 DNA ArtificialSequence Description of Artificial Sequence primer 5 gacactatag aatagggc18 6 51 DNA Artificial Sequence Description of Artificial Sequenceprimer 6 tgggatcatc agcaggacca tgaaacctgc ccaggccaca gaccgcacca t 51 740 DNA Artificial Sequence Description of Artificial Sequence primer 7tacatctaga attatggcaa aactggtctg ctacttcacc 40 8 34 DNA ArtificialSequence Description of Artificial Sequence primer 8 agatctaaccttaggtgcct gaagacaagt atgg 34 9 29 DNA Artificial Sequence Descriptionof Artificial Sequence primer 9 tacagaattc ttattcacat ccggccctg 29 10 34DNA Artificial Sequence Description of Artificial Sequence primer 10tacatctaga ctccatccag aaaaacaggt atgg 34 11 30 DNA Artificial SequenceDescription of Artificial Sequence primer 11 tctagagtcg acctgcaggcatgcaagctt 30 12 50 DNA Artificial Sequence Description of ArtificialSequence primer 12 cgcaagcttg agagctccgt tccgccacat ggtgcggtctgtggcctggg 50 13 32 DNA Artificial Sequence Description of ArtificialSequence primer 13 gactctagac taggtgcctg aaggcaagta tg 32 14 373 PRTHomo sapiens 14 Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala Gln Tyr ArgGln Gly 1 5 10 15 Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro Ser LeuCys Thr His 20 25 30 Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His Gln LeuSer Thr Thr 35 40 45 Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn GlyLeu Lys Lys 50 55 60 Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile Gly GlyTrp Asn Phe 65 70 75 80 Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr AlaAsn Asn Arg Gln 85 90 95 Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg LysTyr Ser Phe Asp 100 105 110 Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly SerGln Gly Ser Pro Ala 115 120 125 Val Asp Lys Glu Arg Phe Thr Thr Leu ValGln Asp Leu Ala Asn Ala 130 135 140 Phe Gln Gln Glu Ala Gln Thr Ser GlyLys Glu Arg Leu Leu Leu Ser 145 150 155 160 Ala Ala Val Pro Ala Gly GlnThr Tyr Val Asp Ala Gly Tyr Glu Val 165 170 175 Asp Lys Ile Ala Gln AsnLeu Asp Phe Val Asn Leu Met Ala Tyr Asp 180 185 190 Phe His Gly Ser TrpGlu Lys Val Thr Gly His Asn Ser Pro Leu Tyr 195 200 205 Lys Arg Gln GluGlu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp Ala 210 215 220 Ala Val GlnGln Trp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile 225 230 235 240 LeuGly Met Pro Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser 245 250 255Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro 260 265270 Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp 275280 285 Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile290 295 300 Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp Val Glu Ser PheLys 305 310 315 320 Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu Gly GlyAla Met Val 325 330 335 Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe SerCys Asn Gln Gly 340 345 350 Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln GluLeu Ser Leu Pro Tyr 355 360 365 Leu Pro Ser Gly Thr 370 15 373 PRT Homosapiens 15 Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala Gln Tyr Arg GlnGly 1 5 10 15 Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro Ser Leu CysThr His 20 25 30 Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His Gln Leu SerThr Thr 35 40 45 Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn Gly LeuLys Lys 50 55 60 Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile Gly Gly TrpAsn Phe 65 70 75 80 Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr Ala AsnAsn Arg Gln 85 90 95 Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg Lys TyrSer Phe Asp 100 105 110 Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser GlnGly Ser Pro Ala 115 120 125 Val Asp Lys Glu Arg Phe Thr Thr Leu Val GlnAsp Leu Ala Asn Ala 130 135 140 Phe Gln Gln Glu Ala Gln Thr Ser Gly LysGlu Arg Leu Leu Leu Ser 145 150 155 160 Ala Ala Val Pro Ala Gly Gln ThrTyr Val Asp Ala Gly Tyr Glu Val 165 170 175 Asp Lys Ile Ala Gln Asn LeuAsp Phe Val Asn Leu Met Ala Tyr Asp 180 185 190 Phe His Gly Ser Trp GluLys Val Thr Gly His Asn Ser Pro Leu Tyr 195 200 205 Lys Arg Gln Glu GluSer Gly Ala Ala Ala Ser Leu Asn Val Asp Ala 210 215 220 Ala Val Gln GlnTrp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile 225 230 235 240 Leu GlyMet Pro Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser 245 250 255 AspThr Arg Val Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro 260 265 270Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp 275 280285 Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile 290295 300 Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys305 310 315 320 Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu Gly Gly AlaMet Val 325 330 335 Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe Ser CysAsn Gln Gly 340 345 350 Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu LeuSer Leu Pro Tyr 355 360 365 Leu Ser Ser Gly Thr 370 16 28 DNA ArtificialSequence Description of Artificial Sequence primer 16 tgatacggtaccgccccatg gctgacta 28 17 20 DNA Artificial Sequence Description ofArtificial Sequence primer 17 gcaagtttgg cgcgaaatcg 20 18 66 DNAArtificial Sequence Description of Artificial Sequence primer 18gcttaagctt gctgcagcct gccgctgagc tgcatcatgc tactactact gctgctgctg 60ggcctg 66 19 115 DNA Artificial Sequence Description of ArtificialSequence primer 19 aacagggccc ttaattaatt aggtacctgc gcggccgcagcatcgattgc tctagaagcg 60 atatcagcga attctgtctg ctcgaagcgg ccggccgccccgactcgaga gtaac 115 20 34 DNA Artificial Sequence Description ofArtificial Sequence primer 20 tatagaattc ttctcctgca accagggccg atac 3421 35 DNA Artificial Sequence Description of Artificial Sequence primer21 tatagaattc ccagagcttg aagttccaaa accag 35 22 35 DNA ArtificialSequence Description of Artificial Sequence primer 22 tatagaattcagccctggac aagacacgtt ctgcc 35 23 35 DNA Artificial Sequence Descriptionof Artificial Sequence primer 23 agaggaattc cagggcaaag ctgatgggct ctatc35 24 37 DNA Artificial Sequence Description of Artificial Sequenceprimer 24 acaggaattc aatcctcggg aacggtccag cttctac 37 25 37 DNAArtificial Sequence Description of Artificial Sequence primer 25cacatctaga ttatgtcggg cagctttgct ggaacag 37 26 36 DNA ArtificialSequence Description of Artificial Sequence primer 26 agagtctagatcaattccag gtgcagcatt tgcagg 36 27 35 DNA Artificial SequenceDescription of Artificial Sequence primer 27 agaggaattc agccctggacaagacacgtt cagcc 35 28 97 DNA Artificial Sequence Description ofArtificial Sequence primer 28 agaggaattc agccctggac aagacacgttctgccagggc aaagctgatg ggctctatcc 60 caatcctcgg gaacggtcca gcttctacagcagtgca 97 29 67 DNA Artificial Sequence Description of ArtificialSequence primer 29 tgcttctaga ttaattccag gtgcagcatt tgcaggagttgctgaacacc aggcctgtcg 60 ggctgct 67 30 36 DNA Artificial SequenceDescription of Artificial Sequence primer 30 tgcttctaga ttaattccaggtgcagcatt tgctgg 36 31 36 DNA Artificial Sequence Description ofArtificial Sequence primer 31 tgcttctaga ttaattccag gtgcagcttt tgcagg 3632 36 DNA Artificial Sequence Description of Artificial Sequence primer32 tgcttctaga ttaattccag gtgctgcatt tgcagg 36 33 72 DNA ArtificialSequence Description of Artificial Sequence primer 33 ctctggtacctttggataaa agagactaca aggacgacga tgacaagagc cctggacaag 60 acacgttctg cc72 34 77 DNA Artificial Sequence Description of Artificial Sequenceprimer 34 atatgcggcc gcgacttatc cactactatg atgatgatga tgatgtcctgctccattcca 60 ggtgcagcat ttgcagg 77 35 37 DNA Artificial SequenceDescription of Artificial Sequence primer 35 ctctgaattc caagacacgttctgccaggg caaagct 37 36 35 DNA Artificial Sequence Description ofArtificial Sequence primer 36 atatgaattc acgttctgcc agggcaaagc tgatg 3537 41 DNA Artificial Sequence Description of Artificial Sequence primer37 aacatctaga ttaggtgcag catttgcagg agttgctgaa c 41 38 48 DNA ArtificialSequence Description of Artificial Sequence primer 38 aagaggtacctttggataaa agaagccctg gacaagacac gttctgcc 48 39 41 DNA ArtificialSequence Description of Artificial Sequence primer 39 gagagcggccgcgacttaat tccaggtgca gcatttgcag g 41

What is claimed is:
 1. A chitin-binding, chitinase-inactive polypeptidecomprising a chitin-binding fragment of the 54 C-terminal amino acids ofhuman chitinase as set forth in SEQ ID NO:
 2. 2. The polypeptide ofclaim 1 selected from the group consisting of a polypeptide having thesequence of amino acid residues 347 through 445 of SEQ ID NO: 2,polypeptide having the sequence of amino acid residues 374 through 445of SEQ ID NO: 2, a polypeptide having the sequence of amino acidresidues 392 through 445 of SEQ ID NO: 2, a polypeptide having thesequence of amino acid residues 395 through 445 of SEQ ID NO: 2, and apolypeptide having the sequence of amino acid residues 397 through 445of SEQ ID NO:
 2. 3. A polypeptide selected from the group consisting ofpolypeptides having the sequence of amino acid residues X through Y ofSEQ ID NO: 2, wherein X is a consecutive integer from 347 through 397and Y is
 445. 4. A chitin-binding, chitinase-inactive polypeptidecomprising a polypeptide of claim
 3. 5. A fusion protein comprising thepolypeptide of claim 1 fused to a heterologous polypeptide.
 6. Thefusion protein of claim 5 wherein the heterologous polypeptide is anenzyme.
 7. A composition comprising the polypeptide of claim 1 and aphysiologically acceptable diluent.
 8. The composition of claim 7further comprising a non-chitinase anti-fungal agent.
 9. A compositioncomprising the polypeptide of claim 1 or 4 conjugated to an anti-fungalagent.
 10. A method of treating fungal infection comprising the step ofadministering to a subject suffering from fungal infection a compositionaccording to any one of claims 7 through
 9. 11. The method of claim 10further comprising the step of administering to said subject anon-chitinase anti-fungal agent.
 12. A composition comprising thepolypeptide of claim 1 or 4 conjugated to a detectable label.
 13. Thecomposition of claim 12 wherein the detectable label is selected fromthe group consisting of radioisotopes, fluorophores, dyes,electron-dense compounds and enzymes.
 14. A method for determining thepresence of chitin in a sample comprising the steps of: (a) contactingthe sample with the composition of claim 12 and (b) determining theamount of labelled polypeptide bound to chitin.
 15. A kit for diagnosingthe presence of chitin in a sample comprising the composition of claim12.
 16. A purified, isolated polynucleotide encoding the polypeptide ofclaim
 1. 17. The polynucleotide of claim 16 that is DNA.
 18. A vectorcomprising the DNA of claim
 17. 19. A host cell stably transformed ortransfected with the DNA of claim 17 in a manner allowing the expressionin said host cell of a polypeptide encoded by said DNA.
 20. A method forproducing a polypeptide comprising a human chitinase fragment comprisingculturing the host cell of claim 19 in a nutrient medium and isolatingsaid polypeptide from said host cell or said nutrient medium.
 21. Apurified, isolated polypeptide produced by the method of claim
 20. 22. Amonoclonal antibody that specifically binds to an epitope within the 54C-terminal amino acids of human chitinase as set forth in SEQ ID NO: 2.23. The monoclonal antibody of claim 22 that competes with monoclonalantibody 243Q, produced by the hybridoma deposited under ATCC AccessionNo. ______, for binding to a chitin-binding, chitinase-inactive fragmentof human chitinase.
 24. The monoclonal antibody of claim 22 thatcompetes with monoclonal antibody 243M, produced by the hybridomadeposited under ATCC Accession No. ______, for binding to achitin-binding, chitinase-inactive fragment of human chitinase.